BIOLOGY 

UBRARY 

G 


IMMUNITY 


METHODS  OF  DIAGNOSIS  AND  THERAPY 

AND 

THEIR  PRACTICAL  APPLICATION 


CITRON 


GARBAT 


IMMUNITY 

METHODS  OF  DIAGNOSIS  AND  THERAPY 

AND 

THEIR  PRACTICAL  APPLICATION 


BY 

DR.  JULIUS  CITRON 

ASSISTANT  AT  THE  UNIVERSITY  CLINIC  OF  BERLIN,  II  MEDICAL  DIVISION 

TRANSLATED  FROM  THE  GERMAN  AND  EDITED 
'  BY 

A.    L.   GARBAT,   M.   D. 

ASSISTANT  PATHOLOGIST  AND  ADJUNCT  VISITING  PHYSICIAN, 
GERMAN  HOSPITAL,  NEW  YORK 


SECOND  EDITION,  REVISED  AND   ENLARGED 

30  ILLUSTRATIONS 
2  COLORED  PLATES  AND  8  CHARTS 


PHILADELPHIA 
P.    BLAKISTON'S   SON   &   CO. 

1012   WALNUT   STREET 


I 


COPYRIGHT,  1914,  BY  P.  BLAKISTON'S  SON  &  Co. 


THE. MAPLE. PRESS. YORK. PA 


TO 

PROFESSOR  FRIEDR1CH  KRAUS 

AS    EVIDENCE    OF  DUE   HONOR   AND   THANKFULNESS 
THIS   BOOK   IS    DEDICATED 

BY   THE    AUTHOR 

ON   THE    OCCASION   OF   THE   OPENING   OF   THE   NEW 
II   MEDICAL  DIVISION 


3C0085 


PREFACE  TO  THE  SECOND  GERMAN  EDITION. 


The  first  edition  of  "Immunity,  Methods  of  Diagnosis  and  Therapy," 
was,  I  am  glad  to  say,  favorably  received.  The  book  apparently  fulfilled 
a  demand,  as  not  quite  two  years  have  elapsed  and  a  second  edition  is 
required.  An  Italian  translation  by  G.  Volpino  and  an  English  edition 
by  A.  L,  Garbat  have  in  the  meantime  appeared.  A  Spanish  and  a  Rus- 
sian publication  are  in  preparation. 

During  the  last  two  years  certain  problems  in  this  field  have  gained 
greatly  in  importance,  and  therefore  require  more  detailed  consideration. 
Special  chapters  have  been  allotted  to  tumor  studies  and  to  anaphylaxis. 
Probably,  even  this  treatise  on  anaphylaxis  will  by  many  be  considered 
too  short.  Without  underestimating  the  distinct  scientific  value  of  the 
very  numerous  articles  being  written  on  this  subject,  I  feel  that  the 
fundamental  principles  of  anaphylaxis  are  still  too  theoretical  and  the 
practical  application  still  too  limited,  to  warrant  a  more  exhaustive  re- 
view in  a  practical  book  such  as  this. 

Chemotherapy,  on  the  other  hand,  has  attained  such  striking  clinical 
importance,  that  I  have  dwelt  in  length  upon  its  development.  As  ever, 
the  practical  elements  are  emphasized.  My  broad  experience  with 
salvarsan  has  aided  me  in  distinguishing  the  points  of  importance  for  the 
practitioner. 

'  Slight  changes  and  additions  have  been  scattered  throughout  the  book. 

I  hope  that  this  second  edition  will  add  new  friends  and  retain  the 
old  ones. 

JULIUS  CITRON. 


Vll 


PREFACE  TO  THE  SECOND  ENGLISH  EDITION. 


The  necessity  for  a  second  English  edition  has  arisen  after  an  interval 
of  a  year.  It  is  a  source  of  satisfaction  to  note  the  apparent  general 
approval  and  demand  which  this  small  book  on  " Immunity"  has  fulfilled. 
The  credit  to  Dr.  Citron  is  well  deserving.  The  text  of  the  new  English 
publication  has  been  carefully  reviewed  and  minor  changes  made,  but  it 
follows  closely  that  of  the  German  with  its  important  additions.  Like 
in  the  first  English  edition,  however,  so  also  in  the  second,  topics  of  more 
recent  development,  treated  only  very  slightly  or  not  at  all  in  the  German, 
have  been  enlarged  or  inserted:  gonococcus  and  typhoid  complement 
fixation  tests,  agglutination  and  hemolysis  tests  for  transfusion,  prophy- 
lactic typhoid  inoculation,  etc.  Friendly  criticism  to  discuss  some  sub- 
jects with  more  detail  has  been  gladly  met  and  corrected,  so  that  a  be- 
ginner may  have  no  greater  difficulty  than  is  only  natural.  It  is  hoped 
that  this  English  edition  will  meet  with  the  same  approbation  which  I 
am  happy  to  say  has  been  accorded  the  former  one. 

A.  L.  GARBAT. 


IX 


PREFACE  TO  THE  GERMAN  EDITION. 


This  book  is  to  serve  a  purely  practical  purpose.  The  methods  of 
serum  diagnosis,  on  account  of  their  growing  clinical  significance,  are 
constantly  stimulating  greater  interest  in  all  branches  of  medical  science. 
While  giving  instruction  in  this  subject,  I  realized  that  it  would  be  of  great 
help  to  both  the  medical  student  and  physician  if  they  possessed  a  short 
text-book  which  would  review  in  a  purely  critical  form  the  various  methods 
of  immunity  diagnosis,  especially  those  relating  to  tuberculosis  and 
syphilis. 

The  two  systems  of  Kolle  and  Wassermann,  and  R.  Kraus  and  Levaditi 
are  doubtless  the  standards  on  the  subject  in  German  medical  literature. 
On  account  of  their  size  and  price,  however,  these  volumes  come  to  be 
sought  only  by  the  specialist. 

It  was  therefore  my  aim  in  this  book  so  to  present  the  subject  of  immu- 
nity that  the  general  medical  man,  who  is  even  slightly  acquainted  with 
laboratory  work,  can  learn  the  details  of  the  various  reactions  and  their 
significance.  In  selecting  the  different  methods,  I  have  taken  up  those 
which  are  used  in  the  clinic  for  diagnostic,  therapeutic,  or  prophylactic 
purposes.  In  addition  there  are  herein  included  certain  fundamental 
considerations  of  questions  on  immunity  which  for  the  present  are  only  of 
theoretical  interest,  but  which  owing  to  the  rapid  development  of  the 
subject,  may  soon  become  of  practical  importance. 

I  have  endeavored  especially  to  place  before  the  reader  a  critical  review 
of  the  results  of  the  various  methods.  In  the  description  of  technical 
details,  the  original  articles  of  the  author  have  been  selected;  modifica- 
tions having  been  considered,  only  provided  they  exhibit  distinct  ad- 
vantages over  the  original  method. 

I  here  wish  to  express  my  thanks  to  my  teacher  Prof.  Wassermann, 
under  whose  guidance  and  stimulus  I  gained  my  laboratory  experience; 
also  to  my  chief  Prof.  Kraus  whose  clinical  genius  proved  to  me  the  practi- 
cal importance  that  this  subject  of  immunity  commands. 

To  the  publishers  as  well,  whose  kind  cooperation  in  all  my  plans  as 
regards  publication  and  illustration,  greatly  simplified  my  work,  I  extend 
my  heartiest  appreciation. 

JULIUS  CITRON. 


XI 


NOTE  BY  THE  AMERICAN  EDITOR. 


The  study  of  "  Immunity,"  once  of  merely  theoretical  interest  and 
purely  scientific  importance,  is  to-day  no  longer  such.  A  realm  of  prac- 
tical considerations,  considerations  which  are  constantly  coming  up  and 
enlisting  the  attention  of  the  busy  practitioner  have  little  by  little  sup- 
planted those  phenomena  at  one  time  vaguely  understood  and  mostly 
taken  for  granted.  Gradually  have  the  uncertainties  so  long  dominating 
and  obscuring  an  intelligent  comprehension  of  the  subject  been  cleared 
away;  mistakes  explained;  and  hypotheses  re-established  as  proven 
facts.  The  methods  employed  for  the  necessary  investigations  have 
naturally  improved  and  increased  with  such  extreme  rapidity  that  a 
severe  task  presents  itself  to  one  who  desires  to  separate  the  more  from 
the  less  valuable  ones.  It  was  therefore  with  extreme  satisfaction  that 
I  greeted  the  opportunity  of  bringing  out  an  English  edition  of  this  work- 
ing hand-book  on  the  various,  but  most  essential  methods  used  in  the 
applications  of  •"  Immunity."  The  author  of  this  volume  has,  by  his 
exhaustive  research  and  extensive  practical  experience  as  a  teacher, 
treated  his  field  with  such  fulness  and  preciseness  of  detail  that  it  is  of 
value  not  only  to  the  laboratory  student,  but  also  to  the  clinician. 

Its  already  favorable  reception  in  Germany  will,  it  is  hoped,  be  ex- 
tended to  it  in  America,  especially  by  those  whose  lack  of  familiarity 
with  the  German  language  has  kept  this  work  beyond  their  reach. 

The  chapter  on  vaccines  has  been  slightly  revised  and  elaborated  to 
conform  more  closely  with  the  most  recently  advocated  methods  of  Sir 
A.  E.  Wright,  to  whom  the  editor  is  indebted  for  his  experience.  Other- 
wise there  has  been  no  need  to  alter  the  original  text,  with  the  exception 
that  here  and  there  some  features  which  may  be  of  special  interest  to 
the  English  reading  public,  have  been  inserted. 

I  wish  in  the  present  connection  to  express  my  deep  thanks  to  my 
teacher  Dr.  Citron  for  offering  me  the  privilege  of  this  undertaking,  and 
to  the  publishers,  Messrs.  Blakiston  &  Co.,  without  whose  hearty  coopera- 
tion this  would  have  been  impossible,  my  sincere  appreciation. 

A.  L.  GARBAT. 


Xlll 


TABLE  OF  CONTENTS. 


CHAPTER  I. 

PAGE 
INTRODUCTION i 

Definitions  of  immunity  and  antibody.  The  law  of  specificity.  The  neces- 
sity of  control  tests. 

CHAPTER  II. 

LABORATORY  EQUIPMENT      . 7 

General  technique.  (Technique  of  injection.  The  methods  of  obtaining  and 
preserving  serum.  Bacterial  filtration.  Dilutions.  Measurement  of  small 
amounts  of  bacteria.) 

CHAPTER  III. 

ACTIVE  IMMUNITY '  ;  -;..v'. V.V*:':V •'•=. '•'-; .-'-.  '..". '•'.- .  ;'.".t'.;. \   .     21 

Immunization  with  living  and  dead  virus.  (Vaccination  against  small-pox; 
antirabic  vaccination;  antityphoid  inoculation.)  :':•%>•: 

CHAPTER  IV. 

ACTIVE  IMMUNITY .    .    ...     36 

Immunization  with  bacterial  extracts.     Aggressin  experiments. 

CHAPTER  V. 

TUBERCULIN  DIAGNOSIS ./'.-..:: ^'^''..  ^:-^'^,  :,\'-,\^'^:\-.  ...     45 

Koch's  method;  cutaneous  reaction;  Moro's  ointment  reaction;  intracutaneous 
reaction;  ophthalmo-reaction;  the  specificity  and  prognostic  value  of  the 
tuberculin  reactions.  Mallein.  Tricophytm. 

CHAPTER  VI. 

TUBERCULIN  THERAPY 60 

The  technique  of  the  tuberculin  therapy;  old  tuberculin;  new  tuberculin; 
bovine  tuberculin.  Nastin. 

CHAPTER  VII. 

TOXIN  AND  ANTITOXIN     .    .    .    .  '.-:. •'••'. •'.'* -• . . :,••* .•: '.'..- .. -^. •>  V-.".   . :. :.-..:.  Xi   .     74 
The  serum  therapy  of  diphtheria. 

CHAPTER  VIII. 

TOXIN  AND  ANTITOXIN  (continued)    .    .  -.    ,  ;.    ,  ^. ',- .    ............     85 

Definition  of  toxin.  Tetanus  toxin.  Botulism  toxin.  Dysentery  toxin. 
Staphylolysin. 

CHAPTER  IX. 

THE  TOXINS  or  THE  HIGHER  PLANTS  AND  ANIMALS  AND  THEIR  ANTIBODIES.     FER- 
MENTS AND  ANTIFERMENTS  . 95 

Snake  poison.     Paroxysmal  hemoglobinuria. 

xv 


XVI  TABLE    OF    CONTENTS 

PAGE 
CHAPTER  X. 

AGGLUTINATION 

Macroscopic    test,     microscopic     test.      Group     agglutination.     Bacterial  105 
agglutinins.     Hemagglutinins.     Transfusion  tests. 

CHAPTER  XI. 

PRECIPITINS 120 

Bacterial  precipitation.     Proteid  precipitation. 

CHAPTER  XII. 

BACTERIOLYSINS  AND  HEMOLYSINS  (CYTOLYSINS) 131 

Technique  of  bacteriolytic  tests.  Pfeiffer's  phenomenon.  Bactericidal  test 
by  plate  method  of  Neisser  and  Wechsberg.  Hemolysins.  Cytolysins. 

CHAPTER  XIII. 

METHOD  OF  COMPLEMENT  FIXATION      152 

Principles  of  this  method.  Antituberculin.  Ehrlich's  side-chain  theory. 
Serum  diagnosis  of  syphilis  and  of  diseases  caused  by  animal  parasites. 

CHAPTER  XIV. 

TECHNIQUE  OF  THE  COMPLEMENT  FIXATION  METHOD 171 

The  original  method  of  Bordet-Gengou.  Wassermann-B ruck's  modification. 
The  technique  of  the  serum  diagnosis  of  syphilis.  Echinococcus  disease. 
Epidemic  meningitis,  tuberculosis,  gonococcus  infections,  typhoid  fever. 
The  differentiation  of  proteids  according  to  Neisser-Sachs. 

CHAPTER  XV. 

PHAGOCYTOSIS.    OPSONINS  AND  BACTERIOTROPINS 196 

Technique  of  opsonic  index  determination  and  of  "Wright's  vaccine  treatment. 
Neufeld's  method  of  examination  for  bacteriotropins. 

CHAPTER  XVI. 

MALIGNANT  TUMORS 216 

Studies  in  reference  to  their  immunity;  serum  reactions;  transplantation 
experiments.  Meiostagmine  reaction. 

CHAPTER  XVII. 

ANAPHYLAXIS 221 

CHAPTER  XVIII. 

PASSIVE  IMMUNITY.     (SERUM  THERAPY) 231 

Bacteriolytic  sera.     Special  serum  therapy. 

CHAPTER  XIX. 

CHEMOTHERAPY 240 

Definition.     Atoxyl.     Salvarsan.     Chemotherapy  of  malignant  tumors. 


LIST  OF  ILLUSTRATIONS  AND  CHARTS. 


FIG.  PAGE 

1.  A  room  in  the  laboratory  of  the  Royal  Institute  for  Infectious  Diseases 

(Berlin) 7 

2.  Standard  for  measuring  the  size  of  platinum  loops  (Czaplewski)    ....  8 

3.  Intravenous  inoculation  (after  Uhlenhuth) .  9 

4.  Intraperitoneal  inoculation  (after  Uhlenhuth) 1 1 

5.  Removal  of  peritoneal  exudate  in  Friedberger's  position  (original)     ...  12 

6.  Veno-puncture  (original) 13 

7.  Wet-cup  method  for  obtaining  blood  (original) 14 

8.  Test-tube  for  preservation  of  serum  (original) 15 

9.  Pukal  filter 16 

10.  Filtration  through  pukal  filter   . 16 

11.  Reichel  filter 17 

12.  Lilliputian  filter 17 

13.  V.  Pirquet's  tuberculin  test  (original)  .    .    ,   ...    .....  Y, :.    .    .    .    .  51 

14.  Ophthalmodiagnosticum  for  tuberculosis  (original) 52 

15-16.  Diagram  for  the  complement  fixation  reaction '.'•;•    •    •    •  154 

17.  Diagram  for  the  complement  fixation  in  syphilis .  .  166 

18-20.  Technique  for  the  determination  of  the  opsonic  index  according  to  Wright  204 

21-22.  Technique  for  the  determination  of  the  opsonic  index  according  to  Wright  204 

23-24.  Technique  for  the  determination  of  the  opsonic  index  according  to  Wright  205 
25-26.  Technique  for  the  determination  of  the  opsonic  index  according  to  Wright  206-7 

27.  Phagocytosis  of  tubercle  bacilli 208 

28-30.  Technique  for  intravenous  injection  of  salvarsan 205-2 

CHART. 

1.  Example  of  a  diagnostic  tuberculin  reaction 48 

2.  Example  of  hypersusceptibility  by  diminution  in  the  tuberculin  dose     .    .  65 

3.  Marked  increase  of  weight  in  a  tuberculous  individual  in  spite  of  con- 

tinued fever  .    .    ....    .    .v   :.'"'..••• 67 

4.  Treatment    with    S.    B.    E.,    almost   without   reaction.    Immunization 

against  B.  E.     .    .   ".    ....•%?,..•.•;! 71 

5.  Opsonic  curve  after  a  small  dose  of  staphylococcus  vaccine     ......  200 

6.  Opsonic  curve  during  treatment  with  new  tuberculin 201 

7.  Increase  in  the  opsonic  index  for  gonococci  by  Bier's  hyperemia    ....  202 

8.  Auto-inoculation  with  tuberculin  after  physical  examination  and  massage  202 


xvii 


IMMUNITY. 


CHAPTER  I. 

INTRODUCTION. — DEFINITIONS  OF  IMMUNITY  AND  ANTIBODY. — THE  LAW 
OF  SPECIFICITY. — THE  NECESSITY  OF  CONTROL  TESTS. 

The  diagnosis  of  infectious  diseases  can  be  approached  in  several  ways. 
In  addition  to  the  aid  obtained  from  clinical  signs  such  as  the  course  of  the 
temperature,  the  changes  in  the  various  organs,  the  exanthemata,  etc.,  the 
finding  of  the  specific  etiological  agent  of  the  disease,  or  the  specific  anti- 
bodies developed  by  the  reaction  of  the  organism,  are  of  equal  or  even 
greater  importance.  •  The  course  of  an  infection  depends  not  only  upon 
the  nature,  the  number,  and  the  virulence  of  the  infecting  agents,  but 
also  upon  the  behavior  of  the  infected  body.  One  must  consider  a  disease 
as  the  result  of  the  interaction  of  both  of  these  factors  without  necessarily 
being  able  to  attribute  the  various  symptoms  to  either  the  one  or  the  other. 
Although  the  general  reaction  of  the  organism  is  varied,  it  can  nevertheless 
be  shown  that  in  spite  of  even  individual  differences,  the  characteristic 
bacteria  and  their  products  bring  about  a  distinct  symptom-complex 
which  is  usually  concomitant  with  a  significant  defense  on  the  part  of 
the  organism.  The  means  which  the  body  employs  in  this  protection 
are  cellular  and  humoral  in  nature.  In  fact,  there  is  a  group  of  infectious 
diseases  in  which  the  cellular  reaction  predominates,  and  another  in  which 
humoral  changes  are  pre-eminent;  and  between  these  extremes  are  various 
intermediate  forms.  Thus,  the  constantly  changing  picture  of  tubercu- 
losis always  shows  the  tubercle  as  its  typical  product  of  cellular  reaction; 
similarly  leprosy  and  syphilis  have  their  peculiar  cellular  changes. 
More  difficult,  however,  to  recognize  by  the  unaided  eye  or  even  the 
microscope,  are  the  finer  biological  alterations  which  take  place  in  the 
body  fluids  during  the  course  of  infectious  diseases.  Here,  special  methods 
are  necessary  to  detect  and  differentiate  the  various  humoral  changes 
which  occur  for  the  main  part  in  the  blood  serum.  As  is  known  at 
present,  the  humoral  as  well  as  the  cellular  immunity  reactions  are  not 
limited  to  infectious  diseases,  but  also  express  normal  physiological  and 
pathological  conditions.  With  the  conception  of  Ehrlich's  side-chain 
theory  the  bridge  of  understanding  for  the  humoral  reaction  was  built,  and 
it  at  once  became  evident  how  the  physiological  phenomena  of  nutrition 
and  production  of  energy  are  identical  in  their  nature  with  processes  which 


.    .  ;    ^**»*      -INTRODUCTION 

*»  *     : 
>   >< 


under  pathological  circumstances  lead  to  the  formation  of  anti-infectious 
bodies.  In  an  analogous  and  no  less  ingenious  manner,  Metchnikoff  has 
shown  that  the  same  cell  group  of  mesenchymal  origin  which  the  organism 
stations  against  bacterial  invasion  has  physiological  and  physio-patho- 
logical functions  to  f  ulfill  in  the  whole  animal  scale.  In  the  lower  animals, 
these  cells  aid  in  the  metamorphosis  of  the  body  structure,  thus  leading 
to  the  disappearance  of  entire  organs.  In  the  female,  they  aid  in  the 
involution  of  the  uterus  after  labor,  while  in  the  aged  they  destroy  the 
nerve  cells  in  the  senile  atrophied  nerve  centers  or  finally  as  chromophages 
turn  the  hair  gray.  The  border-line  between  the  physiological  and  patho- 
logical status  is  biologically  not  sharply  demarcated.  It  is  one  single  chain 
of  manifestations  which  possess  numerous  transitional  phases.  As  the 
methods  of  serum  diagnosis  can  prove  reactions  much  finer  even  than 
those  accomplished  by  chemistry,  their  application  has  not  been  limited 
to  the  chapter  on  infectious  diseases. 

By  their  means  also,  proteids,  even  though  manifest  in  minutest  traces, 
can  be  differentiated.  Similarly,  the  secret  of  blood  relationship  has 
begun  to  be  unravelled;  and  there  is  a  possibility  even  of  solving  the 
problems  of  metaboh'sm. 

Closely  associated  with  serum  diagnosis  is  the  serum  therapy.  Even 
though  the  general  application  of  the  latter  is  not  as  widely  developed  as 
that  of  the  former,  it  must  be  remembered  that  through  this  medium 
diphtheria  has  been  transformed  from  a  fatal  to  a  combatable  disease, 
and  incidentally  made  the  name  of  Behring  immortal.  To-day,  attempts 
are  constantly  being  made  to  treat  other  bacterial  and  toxic  diseases 
by  specific  therapy  and  it  is  to  be  hoped  that  success  will  soon  be  met 
with. 

The  study  of  serum  therapy  and  serum  diagnosis  is  undertaken  in 
various  ways.  It  is  comparatively  simple  to  learn  only  the  purely 
technical  details.  All  large  laboratories  have  trained  assistants  for  the 
performance  of  certain  reactions  or  groups  of  reactions  with  absolute 
precision.  Although  as  we  have  said,  they  do  such  work  as  assigned  to 
them  with  accuracy,  they  are  nevertheless  far  from  a  thorough  understand- 
ing of  the  subject  of  serum  diagnosis.  Unfortunately  this  blind  method 
of  procedure  has  recently  been  advocated  to  an  alarming  extent.  In 
addition,  the  practical  success  which  the  Wassermann  reaction  has  met 
with  has  inculcated  the  desire  in  a  certain  class  of  physicians,  for 
carrying  out  this  test  alone,  and  thus  to  become  independent  of  the 
use  of  large  laboratories.  To  meet  this  demand,  short  courses  have  been 
established  and  the  serum  diagnosis  of  syphilis  taught  with  lightning 
rapidity.  That  such  a  state  of  events  is  absolutely  injurious  is  clearly 
evident.  It  is  impossible  for  one  to  be  a  specialist  in  a  certain  reaction 
and  at  the  same  time  be  ignorant  of  the  other  phases  in  the  study  of 


C  INCEPTION  OF  IMMUNITY  3 

immunity.     Unreliable  and  erroneous  results  are   the  inevitable  out- 
comes of  such  unscientific  work. 

The  plan  followed  in  this  book  consists  in  taking  up  all  of  the  important 
principles  and  methods  of  immunity,  even  though  at  present  some  may 
attract  no  direct  practical  attention.  The  principle  of  the  now  widely 
important  Wassermann  reaction  had  been  described  years  previously  by 
Bordet  and  Gengou,  but  merely  from  a  purely  theoretical  standpoint. 
Only  with  the  development  of  the  Wassermann  test  did  it  attain  its  prac- 
tical importance. 

To  start  systematically,  it  is  necessary,  primarily,  to  under- 
Conception  stand  certain  terms  frequently  employed.     First,  the  word 
of  Immunity,  immunity  requires  explanation: 

After  an  individual  has  recovered  from  an  infectious  disease, 
he  passes  into  a  state  where  he  is  less  or  even  not  at  all  susceptible  to  the 
same  infection,  although  no  macroscopical,  microscopical  or  chemical 
change  can  be  shown  to  have  taken  place  in  his  system.  This  condition  is 
one  of  immunity.  And  as  the  body  itself  by  its  own  struggle  with  the 
invading  bacteria  has  brought  about  this  immunity,  it  is  known  as  "  active 
immunity."  Jenner  and  Pasteur  have  employed  this  mode  of  immunity 
acquired  spontaneously  by  overcoming  an  infection  in  their  principle 
of  prophylactic  vaccination.  The  exact  nature  of  this  active  immunity 
is  only  partially  understood.  It  can  be  shown,  however,  that  the  indi- 
viduals thus  actively  immunized  have  within  their  organism  reaction 
bodies  of  a  specific  nature  directed  against  the  infecting  elements  and 
their  poisonous  products.  These  reaction  bodies,  which  circulate  mainly 
in  the  blood  serum,  are  known  as  Antibodies. 

The  antibodies  are  of  differert  classes  depending  entirely  upon  their 
varied  forms  of  activity.  While  some,  such  as  the  agglutinins  and  pre- 
cipitins,  have  the  property  of  grouping  their  respective  invading  agents 
into  small  clumps  or  precipitates,  without  at  the  same  time  embracing 
protective  powers,  there  are  other  antibodies  which  act  essentially  for 
the  defence  of  the  organism.  They  attain  this  by  neutralizing  the 
poison  of  the  bacteria  (antitoxins)  or  by  destroying  the  bacteria  (bacteri- 
olysins),  or  so  altering  the  bacteria  that  the  latter  can  be  more  easily 
destroyed  by  the  cells  (bacteriotropins,  opsonins).  The  last  three  types 
of  immunity  can  be  designated  respectively,  as  antitoxic,  bactericidal,, 
and  cellular  immunity.  Naturally  there  are  many  intermediate  forms. 
It  is  very  probable  that  besides  these  well-recognized  forms  of  immunity 
there  may  be  others,  still  unknown.  Cellular  immunity  must  surely  have 
a  far  greater  range  of  importance  than  is  at  present  ascribed  to  it.  There 
is,  no  doubt,  a  distinct  cell  immunity  which  acts  without  the  aid  of  any 
serum  substance  and  is  known  as  "Tissue  Immunity"  ("histogene" 
Immunitat) . 


4  INTRODUCTION 

If  the  serum  of  an  animal  which  has  been  immunized,  and  containing 
antibodies,  is  injected  into  another  normal  but  non-immunized  animal, 
the  latter  acquires  the  power  of  being  immune  against  the  specific  infective 
agent.  In  this  case  the  immunity  was  not  established  by  direct  cell 
activity  on  the  part  of  the  animal,  for  the  organism  remained  passive, 
and  had,  as  it  were,  immunity  thrust  upon  it.  This  form  of  immunity 
in  contradistinction  to  "active  immunity"  is  designated  as  "passive 
immunity." 

The  forms  of  immunity  thus  far  mentioned  were  all  "acquired" 
either  by  the  spontaneous  recovery  from  the  infection  or  the  artificial 
transmission  of  the  curative  antibodies.  In  contrast,  however,  to  this 
"acquired"  immunity  there  is  a  "natural"  immunity  by  which  is  under- 
stood that  some  animal  species  are  not  at  all  susceptible  to  certain  infec- 
tions. Thus,  man  has  a  natural  immunity  against  a  group  of  diseases 
markedly  fatal  for  some  of  the  lower  animals,  e.g.,  chicken-cholera  and 
hog-cholera.  That  this  natural  immunity  is  almost  always  cellular  in 
character  is  undeniably  true;  and  the  most  important  form  of  this  natural 
armament  against  infection  is  the  powerful  leucocyte,  capable  of  engulfing 
and  destroying  the  invading  enemy.  In  other  words,  phagocytosis. 

Finally  one  should  speak  of  a  "local"  and  "general"  immunity, 
meaning  to  express  thereby  the  different  resistance  and  susceptibility 
that  various  organs  of  the  same  individual  display;  and  also  of  a  "relative" 
and  "absolute"  immunity  in  order  to  differentiate  quantitatively  a 
transitory  immunity  from  one  that  is  of  long  duration. 

Another  term  very  often  employed  is  "antibody."     This,  as 
has  already  been  explained,  is  a  name  used  to  designate  the 
Antibody      sPecmc  bodies  which  the  organism   produces  -as  a   reaction 
against  the  infecting  agents  and  their  toxic  products.     Anti- 
bodies are  also  formed  when  animals  are  injected  with  foreign  proteids 
not  of  bacteria]  origin,  such  as  the  blood  from  a  different  animal  species, 
egg  albumin,  etc.     In  order  that  these  antibodies  may  be  obtained,  the 
substances  employed  must  enter  the  system  "parenteral,"  i.e.,  some  way 
outside  of  the  gastrointestinal  tract. 

In  older  literature  the  terms  antibody  and  protective  body  were  used 
synonymously.  That  is  decidedly  incorrect,  inasmuch  as  not  all  anti- 
bodies possess  the  power  of  protection  and  not  every  actively  immune 
organism,  demonstrable  antibodies.  Furthermore,  antibodies  as  the 
bacteriolysins  which  are  generally  considered  to  have  protective  powers, 
and  correctly  so  too,  can  exist  in  a  system  in  large  numbers  with  out  neces- 
sarily rendering  that  organism  immune. 

How  complicated  various  chapters  in  the  study  of  immunity  can  be  will  be 
clearly  evidenced  by  a  few  of  the  author's  experiments  with  the  hog-cholera 


THE   LAW   OF    SPECIFICITY  5 

bacillus.  Rabbits  rendered  actively  immune  by  inoculation  with  extracts  of  hog- 
cholera  bacilli  possess  a  serum  which  when  injected  into  an  animal  of  a  different  species, 
as  the  guinea-pig,  will  render  the  latter  passively  'mmune.  If,  however,  the  serum  is 
injected  into  another  animal  of  the  same  class  (another  rabbit),  no  protective  power  is 
transmitted.  In  other  instances  it  was  shown  that  the  rabbit  which  was  being  treated 
with  the  purpose  of  active  immunity  was  in  reality  never  immune,  as  it  always  suc- 
cumbed when  injected  with  living  bacteria  even  though  its  serum  contained  bodies 
which  were  perfectly  able  to  passively  protect  guinea-pigs  against  the  same  deadly 
infection . 


Just  as  it  is  incorrect  to  consider  an  antibody  and  protective  body  as 
one  and  the  same  thing,  it  is  equally  erroneous  to  deny  the  existence  of 
protective  bodies,  because  their  presence  cannot  be  demonstrated  by  a 
certain  method  of  laboratory  examination.  It  must  be  kept  in  mind 
that  there  are  still  many  unsolved  problems  in  the  subject  of  immunity 
and  that  therefore  only  the  positive  findings  should  be  the  basis  for  drawing 
conclusions. 

In  order  to  learn  the  nature  of  these  antibodies  attempts  have  been 
made  to  isolate  them  chemically.  Thus  far  all  such  trials  have  been 
unsuccessful.  It  is  even  uncertain  whether  these  so-called  antibodies  are 
definite  chemical  entities.  Only  the  effects  of  the  serum  as  a  whole  are 
known,  and  the  ingredients  in  it  to  which  these  activities  are  attributed 
are  thought  of  as  antibodies.  For  didactic  purposes  antibodies,  as 
antitoxins,  agglutinins,  etc.,  will  be  spoken  of  in  this  book  when  the  anti- 
toxic or  agglutinating  properties,  exclusively,  are  meant. 

In  spite  of  the  individual  differences  which  are  ascribed  to  the 
The  Law  of  various  classes  of  antibodies,  there  is  one  quality  possessed  by 
Specificity.  all — their  specificity.  To  explain  this  by  a  rather  crude 
example,  may  be  mentioned  the  fact  that  typhoid  antibodies 
will  give  their  various  reactions  of  immunity  only  when  these  are  per- 
formed with  the  typhoid  bacillus,  and  cholera  antibodies  only  when  per- 
formed with  the  cholera  vibrio.  Substances  which  lack  this  essential 
property  of  specificity  cannot  be  considered  antibodies,  although  they 
may  fulfill  all  other  requirements.  There  are  indeed  limitations  to  this 
fast  rule,  but  these  will  be  considered  subsequently.  '  For  the  present 
the  following  can  be  taken  as  a  fixed  fact;  namely,  that  every  true 
antibody  is  absolutely  specific,  and  that  all  substances  or  bodies  which  are 
not  specific  cannot  be  real  antibodies.  The  law  of  specificity  is  the  funda- 
mental principle  of  serum  diagnosis.  As  soon  as  the  specificity  of  a  reac- 
tion becomes  doubtful,  its  diagnostic  importance  suffers  greatly.  In 
the  following  pages,  therefore,  the  question  whether  or  not  a  reaction  is 
specific  will  be  repeatedly  discussed,  and  it  will  be  the  aim  in  every  way 
possible,  especially  by  the  use  of  control  tests  and  experiments  to  outline 
the  limits  of  this  specificity.  Here,  even  at  so  early  a  stage  of  the  discus- 


6  INTRODUCTION 

sion,  the  absolute  necessity  of  these  control  tests  must  be  urged; 
though  it  may  appear  superfluous  to  the  beginner,  that  for  apparently 
simple  experiments,  controls  are  performed  which  consume  more  time 
than  the  actual  diagnostic  test  itself.  Probably  also  the  desire  will 
arise,  and  perhaps  be  satisfied,  to  omit  these  control  experiments.  Not- 
withstanding the  possibility  that  for  a  long  time  perfectly  good  results 
will  be  obtained,  it  cannot  be  too  often  or  too  emphatically  impressed 
upon  all  workers  in  immunity  methods,  that  the  only  guard  against 
mistakes  and  failures  in  diagnosis  is  necessarily  found  in  control  tests. 
And  especially  in  doing  research  work,  the  latter  are  indispensable.  For, 
experimental  work  which  involves  reasonable  possibilities,  or  has  any 
pretension  towards  plausibility,  warrants  no  true  scientific  conclusion 
without  the  employment  of  such  tests. 

The  author  has  made  it  a  rule,  whenever  new  findings  in  serum  diagnosis  are 
published,  always  to  look  for  the  given  control  experiments.  If  these  are  insufficient, 
then  no  matter  what  the  contents  are,  the  value  of  the  research  is  slight,  for  all 
its  claims  only  may,  but  not  necessarily  must,  be  correct. 


CHAPTER  II. 
LABORATORY  EQUIPMENT. — GENERAL  TECHNIQUE. 

Although  some  of  the  tests  of  serum  diagnosis  are  comparatively 
simple  and  can  be  performed  in  one's  office  or  even  at  the  bedside,  in 
most  instances  a  laboratory  equipment  is  essential.  This  of  course  does 
not  at  all  imply  the  necessity  of  such  elaborate  apparatus  as  one  is  accus- 
tomed to  find  in  our  present  up-to-date  bacteriological  or  serological 


FIG.  i. — A  room  in  the  laboratory  of  the  Royal  Institute  of  Berlin  for  the  study  of  infectious 

diseases. 

laboratories  where  a  great  deal  of  complicated  research  work  is  done. 
For  the  practical  application  of  serum  diagnosis,  as  employed  at  the  hos- 
pital or  in  private  practice,  an  outfit  much  less  costly  is  perfectly  suffi- 
cient. As  regards  the  question  of  a  room,  the  selection  of  one  with  two 
windows,  allowing  the  entrance  of  sufficient  light,  is  indispensable.  At 
the  same  time,  however,  some  arrangement  should  be  made  in  connec- 
tion with  the  windows  in  order  that  the  direct  rays  of  the  sun  be  prevented 
from  striking  one's  desk.  Strong  sunlight  may  weaken  or  even  destroy 

7 


O  LABORATORY  EQUIPMENT 

the  virulence  of  cultures,  or  bring  about  many  changes  in  sera.  Even 
diffuse  daylight  should  not  be  considered  as  entirely  inert.  A  general 
rule  to  be  remembered  is  never  to  expose  any  biological  reagent,  be  it  a 
bacterial  culture  or  any  form  of  its  derivative,  a  serum,  or  any  other  sub- 
stance to  daylight  any  longer  than  is  absolutely  necessary.  If  this  dictum 
is  followed,  one  will  avoid  many  a  difficulty. 

To  conform  with  this  idea,  it  is  wise  to  have  upon  the  table  a  small  closet  into  which 
the  cultures  and  sera  can  be  placed  for  the  time  that  they  are  being  used.     Such  a  con- 
venient receptacle  can  be  made  out  of  a  large 
cigar  box,  painted  black  inside  and  out,  with  its 
lid  replaced  by  a  small  black  curtain. 

The  table  or  desk  at  which  one  works  should 
be  near  the  window,  and  covered  with  filter-paper, 
upon  which  should  come  a  glass  or  asbestos  plate. 
Instead  of  a  wooden  table  it  is  certainly  more 
elegant,  but  costlier  to  have  a  top  plate  of  glass. 
Upon  the  table  there  should  be  a  Bunsen  burner, 
a  microscope,  a  lamp  for  microscopic  work  at 
night,  a  dish  filled  with  sublimate  or  cresol  into 
which  the  infected  substances,  old  cultures,  used 
pipettes  and  graduates  are  placed. 

It  is  very  convenient  to  have  running  water 
and  a  hood  in  the  same  room.  Still  neither  of 
these  is  absolutely  necessary.  As  for  larger  appa- 
ratus— must  be  mentioned,  a  thermostat,  a  mechanism  for  shaking,  a  dry  sterilizer, 
a  good  autoclave,  a  water-bath,  an  instrument  sterilizer,  a  water  or  electrical  cent- 
rifuge, an  ice  chest,  a  closet  for  instruments  and  glassware,  and  finally  animal  cages 
of  the  kind  that  are  easily  cleansed. 

As  for  instruments  and  glassware  the  following  are  required:  scalpels,  scissors, 
forceps,  glass-cutter,  sterilizable  syringes  of  various  sizes,  graduates  of  10,  25,  100,  and 
1000  c.cm.  capacity  each,  pipettes  of  i  c.cm.  with  i/ioo  divisions  and  pipettes  of  10 
c.cm.  with  i/io  divisions,  a  sterilizable  pipette  retainer,  Erlenmeyer  flasks,  Petri  and 
KolleV  dishes,  test-tubes,  dark  glass  flasks,  ordinary  water  glasses,  funnels,  glass  tubing 
of  various  sizes,  and  test-tube  racks.  Furthermore,  a  platinum  needle  and  a  platinum 
loop  are  required.  For  making  a  loop  of  a  definite  size,  and  one  which  can  always  be 
referred  to,  the  small  instrument  devised  by  Czaplewski  is  of  great  help.  It  consists 
of  four  round  metal  bars  i,  2,  3,  and  5  mm.  in  diameter  around  which  the  platinum 
wire  can  be  twisted  in  order  to  make  a  standard  loop  (Fig.  2). 

All  instruments  and  glassware  used  for  serum  work  should  be  per- 
fectly clean.  It  is  best  to  have  all  the  glassware  plugged  with  non- 
absorbent  cotton,  and  sterilized  by  dry  heat.  It  is  never  advisable  to 
clean  the  glassware  with  strong  acids,  alkalies  or  other  strong  chemicals. 
If  this  has  been  done,  the  chemicals  must  be  thoroughly  removed  by  wash- 
ing, as  the  slightest  trace  may  interfere  with  the  accuracy  of  some  tests. 

All  used  glassware  should  at  once  be  placed  into  a  disinfecting  solution. 
For  this  purpose,  lysol,  lysoform  and  cresol  solutions  are  highly  recom- 


FIG.  2. — Instrument  (After  Cza- 
plewski) for  the  standardization  of 
platinum  loops. 


TECHNIQUE    OF   INOCULATION  9 

mendable.  Sublimate  is  less  efficient  because  it  coagulates  albumins  and 
thus  may  lead  to  plugging  of  pipettes  which  may  have  contained  blood 
rests.  If  highly  infectious  material  has  been  examined,  it  is  best  to  place 
the  entire  disinfectant  solution  containing  the  used  glassware  into  the  auto- 
clave, sterilize  it  there,  then  wash  the  supply  thoroughly  with  soap,  dry 
and  resterilize  it  by  dry  heat  for  i  to  2  hours  at  120°  C. 

The  Technique  of  Inoculation. 

Both  for  serum  diagnosis  and  serum  therapy,  the  serum  is  required 
from  animals  which  have  been  artificially  immunized  against  the  bacteria 
or  their  products  of  secretion.  Almost  without  exception,  this  immuni- 
zation is  produced  by  injecting  the  animal  with  the  infectious  virus.  The 
method  of  inoculation  is  either  intravenous,  intraperitoneal,  or  subcutaneous. 

The  technique  of  intravenous  injection  varies  somewhat  with 
Intravenous  different  animals.  In  rabbits,  the  veins  running  along  the 
Injection.  outer  margins  of  the  ears  are  most  suitable.  The  assistant 

sits  upon  a  chair,  holds  the  hind  legs  and  body  of  the  rabbit 
tightly  fixed  between  his  knees  and  thus  has  his  hands  free  to  steady  the 
rabbit's  ears.  Another  method  consists  in  placing  the  rabbit  upon  the 


FIG.  3. — Intravenous  inoculation.     (After  Uhlenhuth.) 

table  and  firmly  holding  him  there  while  the  injection  is  made  (Fig.  3). 
The  ear  is  first  struck  gently  with  the  fingers  and  washed  with  alcohol  and 
xylol.  If  the  hair  is  very  long,  it  should  be  clipped.  If  the  vein  running 
along  the  outer  margin  of  the  ear  is  exceptionally  small,  it  can  be  made 
more  prominent  by  compressing  it  between  the  thumb  and  index  finger 
at  the  root  of  the  ear.  No  force  should  be  used  with  the  injections;  the 
fluids  should  be  allowed  to  flow  into  the  blood  stream  very  slowly.  Glass 


10  LABORATORY  EQUIPMENT 

syringes,  or  such  as  can  be  sterilized  easily,  are  preferable.  Air  bubbles 
are  to  be  carefully  guarded  against  in  order  to  exclude  the  danger  from 
air  embolism. 

If  infectious  material  is  used  for  injection,  it  is  advisable  in  such 
instances'  to  place  a  small  piece  of  cotton  moistened  in  alcohol  or  a  5 
per  cent,  carbolic  acid  solution  around  the  point  of  union  between  the 
needle  and  the  barrel  of  the  syringe  to  prevent  the  possible  escape  of  any 
fluid  which  usually  occurs  at  this  point. 

After  inoculation  is  completed,  the  needle  should  be  quickly  with- 
drawn, a  small  piece  of  non-absorbent  cotton  placed  upon  the  needle 
puncture  and  compression  applied.  If  non-virulent  bacteria  or  albumin 
is  injected,  the  bleeding  may  be  almost  instantly  controlled  by  firmly 
squeezing  the  vessel  above  the  puncture  wound  with  the  edge  of  one's 
finger  nail. 

In  guinea-pigs  intravenous  inoculation  is  more  difficult,  as  here  there 
are  no  large  superficial  veins.  The  Jugular  or  Iliac  vein  is  therefore 
chosen,  and  must  be  dissected  free.  It  is  not  necessary  to  tie  off  the  vessel, 
but  the  wound  should  be  firmly  compressed  by  means  of  clean  gauze  or 
cotton.  Morgenroth  has  substituted  the  simpler  method  of 
Intracardial  intracardial  inoculation.  The  point  of  maximum  pulsation  of 
Injection,  the  heart  to  the  left  of  the  sternum  is  made  out  by  palpation 
and  a  thin  sharp  needle  is  inserted  at  the  specified  area.  The 
spurting  of  blood  indicates  that  the  needle  is  within  the  heart.  Thereupon 
the  already  filled  syringe  is  carefully  fitted  on  to  the  needle  and  the  con- 
tents slowly  injected.  The  syringe  is  then  detached  from  the  needle  and 
blood  is  again  allowed  to  spurt  out  in  order  to  be  absolutely  convinced 
that  the  needle  was  still  in  the  heart.  It  is  next  quickly  withdrawn.  By 
this  method  it  is  possible  to  inject  about  1 1/2  c.cm.  directly  into  the  blood 
stream. 

In  dogs,  sheep,  goats,  horses,  etc.,  the  intravenous  injection  is  given  into  the  jugular 
vein  directly  through  the  skin  which  must  be  thoroughly  shaved,  cleaned  and  dis- 
.infected.  Compression  by  the  finger  makes  the  vein  more  prominent. 

In  dogs  the  popliteal  vein  is  frequently  selected.  In  man  the  intravenous  injection 
is  given  into  one  of  the  veins  on  the  anterior  surface  of  the  elbow  joint. 

Several  general  rules  are  to  be  observed  when  giving  intravenous  in- 
oculations. First  of  all,  only  small  quantities  of  fluids  should  be  injected; 
secondly,  the  temperature  of  the  fluids  for  injection  should  not  differ  from 
that  of  the  body;  thirdly,  substances  that  are  strongly  hemolytic  may  pro- 
duce marked  disturbances  or  even  sudden  death  of  the  animal;  fourthly, 
if  an  animal  is  to  be  frequently  inoculated  it  is  best  to  puncture  the  vein 
for  the  first  inoculations  as  far  peripherally  as  possible  and  give  each  subse- 
quent injection  more  centrally,  for  very  often  thrombi  are  formed  at  the 
site  of  inoculation. 


INTRAPERITONEAL   INJECTION 


II 


INTRAPERITONEAL   injection    is    employed    most    frequently 

Intraperito-  among  rabbits  and  guinea-pigs.     The  main  danger  associated 

neal        with  this  method  is  possible  injury  to  the  intestines;  but  by 

Injection,     heeding  the  following  advice,  this  can  be  prevented.     The 

animal  should  be  fixed  or  held  head  down.  In  this  position, 
the  loops  of  intestines  tend  to  sink  toward  the  diaphragm.  This  is 
further  helped  along  by  gentle  downward  massage  over  the  abdomen 
thus  leaving  an  area,  above  the  bladder,  which  is  sometimes  free  from 
intestines.  Another  protective 
measure  consists  in  using  a  blunt 
canula  which  can  be  made  by 
breaking  off  the  sharp  point  of  the 
needle.  As  it  is  at  times  difficult 
to  pierce  the  skin  with  this  blunt 
instrument,  it  is  advisable  to  pre- 
viously make  a  minute  incision 
through  the  cutis  and  subcutis 
with  a  sharp  pair  of  scissors  and 
pass  the  needle  through  this  small 
opening.  The  needle  should  not 
be  plunged  directly  into  the  peri- 
toneal cavity,  because  at  the  with- 
drawal, the  injected  fluid  easily 
escapes  through  the  punctured 
opening.  First,  it  is  inserted  sub- 
cutaneously  upward,  in  the  long  di- 
rection of  the  animal ;  then  the  hand 
is  raised  and  the  needle  forced 
horizontally  forward  through  the 
peritoneum,  thus  leaving  the  open- 
ing in  the  peritoneum  at  a 
different  level  than  the  one  through  the  muscles  and  fascia,  thereby 
making  the  escape  of  fluid  more  difficult.  One  readily  realizes  that 
he  has  gone  through  the  peritoneum  by  a  relaxation  of  the  reflex  ab- 
dominal rigidity  (Fig.  4) . 

For  the  intraperitoneal  injection  in  guinea-pigs,  Friedberger  has  devised 
a  procedure  which  is  very  satisfactory  and  furthermore  does  away  with 
the  necessity  of  an  assistant.  It  can  also  be  employed  in  Pfeiffer's  test 
for  the  removal  of  exudates  from  the  peritoneal  cavity.  The  guinea-pig 
is  allowed  to  creep  into  the  breast  pocket  of  the  laboratory  gown  until 
its  head  and  thorax  are  inside  of  the  pocket.  Its  hind  legs  are  grasped 
between  the  middle  and  ring  fingers  of  the  left  hand  and  flexed  on  its  back, 
thus  giving  a  free  exposure  of  the  lower  parts  of  the  abdomen  (Fig.  5). 


FIG.  4. — Intraperitoneal  injection  of  rabbit. 
(After  Uhlenhuth.) 


12 


LABORATORY  EQUIPMENT 


SUBCUTANEOUS  inoculation  is  the  simplest  of  all  methods. 
A  fold  of  skin  is  elevated  between  the  thumb  and  index  finger 
°*  tne  ^e^  nan<^  an(^  tne  needle  plunged  into  the  subcutaneous 
tissue.  In  rabbits  and  guinea-pigs  the  skin  of  the  back  or 
abdomen  is  chosen,  as  the  subcutaneous  tissue  here  is  not  tense.  In  goats, 
sheep,  and  horses  the  skin  of  the  neck  and  shoulder  region  is  preferred. 


Sub- 
cutaneous 
Injection. 


FIG.  5. — Removal  of  peritoneal  exudate.    Guinea-pig  held  in  Friedberger's  position.    (Original.') 

The  skin  of  the  back  and  abdomen  is  to  be  avoided  because  following  the 
injection  edema  frequently  arises,  which  may  extend  to  the  lower  extrem- 
ities and  thus  interfere  with  locomotion. 

If  abscesses  arise  after  subcutaneous  injection,  they  should  be  opened, 
washed  out  with  lysol  solution  and  dressed  with  iodoform  gauze. 

The  Methods  of  Obtaining  and  Preserving  Serum. 

Venesection  or  venous  puncture  is  the  method  best  adapted  for  obtain- 
ing blood  from  animals.     The  veins  employed  for  that  purpose  are  those 


OBTAINING  BLOOD    IN   MAN 


which  have  already  been  mentioned  in  connection  with  intravenous  in- 
jections. A  simple,  large,  hollow  needle  is  all  that  is  required.  Suction 
with  a  syringe  is  superfluous.  Only  in  Morgenroth's  method  of  removing 
blood  directly  from  the  heart  of  guinea-pigs  is  aspiration  necessary.  From 
rabbits  enough  blood  can  be  collected  by  making  an  incision  into  the  vein 
along  its  long  axis  with  a  sharp  knife,  or  by  dividing  the  vein  transversely 
with  the  scissors.  The  blood  thus  collected  is  not  absolutely  sterile. 

In  man,  if  only  a  small  quantity  of  blood  is  required,  it  can  be  obtained 
from  the  finger  or  ear.  If,  however,  a  larger  amount  is  necessary,  puncture 
of  one  of  the  veins  in  the  bend 
of  the  elbow  with  the  Strauss 
canula  is  resorted  to.  It  goes 
without  saying  that  this  area 
must  be  properly  disinfected 
with  soap  and  water,  ether, 
alcohol  or  sublimate  solution. 
Wright's  method  for  collect- 
ing moderate  quantities  of 
blood  will  be  reviewed  in  the 
chapter  on  opsonic  studies. 

If  the  vein  is  prominent, 
the  canula  is  thrust  into  the 
vein  directly  through  the  skin. 
Here  the  author  has  found  it 
more  convenient  to  point  the 
canula  upward,  i.e.,  in  the 
direction  of  the  blood  stream.1 
In  cases  where  the  vein  does 
not  stand  out  it  can  be  made 
to  do  so  either  by  applying 
pressure  with  the  finger  upon  its  central  part  or  placing  a  tight  rubber 
bandage  or  rubber  tube  about  the  arm.  This  should  not,  however,  be 
tight  enough  to  obliterate  the  radial  pulse.  In  very  fat  individuals, 
occasionally  even  these  means  may  not  suffice  so  that  the  vein  must  be 
dissected  free  and  incised.  After  completion  of  the  venesection  the  arm 
is  elevated,  slight  pressure  made  upon  the  wound  with  sterile  cotton 
and  a  bandage  applied.  If  a  small  amount  of  blood  is  sufficient,  and, 
as  in  most  serological  examinations  absolute  sterility  is 
not  essential,  venesection  can  be  replaced  by  the  method  of 


FIG.  6. — Puncture  of  vein.     (Original.) 


Wet-cupping. 


wet-cupping.     For  this  procedure  a  scarifier  and  Bier  cup 
are  required.     The  technique  is  as  follows  (Fig.  7). 


1  The  editor  has  found  that  more  blood  is  obtained  by  thrusting  the  canula  into  the  vein  in 
the  reverse  direction. 


14  LABORATORY  EQUIPMENT 

Some  part  of  the  skin  of  the  back  is  thoroughly  disinfected  and  a  well-fitting  Bier 
cup  firmly  applied.  Suction  arises  and  the  skin  assumes  a  dark  bluish-red  appearance. 
After  half  a  minute  the  cup  is  removed,  the  scarifier  applied  and  the  cutting  edges  set 
free.  The  scarifier  is  then  reapplied,  but  this  time  at  right  angles  to  the  previous 
incisions  and  the  edges  again  set  free.  Suction  is  again  made  by  the  Bier  cup  and  the 
blood  is  thus  forced  out  from  the  multiple  incisions. 

The  blood  obtained  by  any  of  the  above  methods  is  collected  into  a 
sterile  vessel  (graduate,  flask,  test-tube)  and  allowed  to  coagulate.  The 
clot  is  then  separated  from  the  sides  of  the  vessel  by  a  sterile  glass  rod  or 
platinum  needle,  the  vessel  plugged  with  absorbent  cotton  and  placed  into 
the  ice  chest.  After  12  to  24  hours  the  serum  begins  to  separate  out  from 


FIG.  7. — Obtaining  blood  by  the  wet-cupping  method. 

the  clot.  If  the  serum  is  required  immediately,  the  blood  is  allowed  to  flow 
directly  into  centrifuge  tubes,  the  clot  separated  from  the  sides  and  the 
tubes  centrifugalized.  With  a  well-regulated  centrifuge  serum  appears 
after  several  minutes. 


There  are  several  rules  to  be  kept  in  mind  when  using  a  centrifuge. 
Rules  for     i.  The  machine  must  be  well  oiled. 

the  Use  of    2.  The  counterbalance  must  be  absolutely  of  the  same  weight. 
Centrifuge.  3.  The  centrifuge  should  never  be  suddenly  stopped,  but  allowed  to  do 

so  of  its  own  accord. 

4.  In  starting  it,  the  motor  should  be  gradually  turned  on. 

5.  If  the  centrifuge  is  slightly  out  of  order  it  should  not  be  used,  but  repaired  at 
once,  otherwise  it  may  be  ruined  forever. 

6.  One  should  never  centrifugalize  with  cotton  plugs  in  the  test-tubes.     If  the 
latter  must  be  sealed,  rubber  stoppers  should  be  used. 


METHOD   FOR   PRESERVING   A    SERUM  15 

The  color  of  a  serum  is  greatly  variable,  depending  mainly  upon  its 
hemoglobin  or  fat  content.  Blood  taken  at  the  height  of  the  period  of 
digestion  shows  a  chylous  serum.  The  serum  of  nursing  women  contains 
milk,  that  of  icteric  people  contains  bile.  For  most  serological  examina- 
tions these  elements  in  the  serum  are  inert,  and  do  not  interfere  with  the 
reading  of  the  results.  In  precipitin  reactions,  however,  the  serum  must 
be  absolutely  clear. 

If  serum  is  to  be  kept  for  a  long  time,  there  are  several  ways  that  it  may 
be  retained  without  losing  its  activity.  The  method  chosen  depends  upon 
the  serum  substance  which  is  to  be  preserved. 

As  will  be  pointed  out  again,  substances  are  either  thermostabile  or 
thermolabile.     The  preservation  of  thermostabile  substances  (agglutinins, 
amboceptors)  is  usually  very  simple.     It  is  sufficient  to  place 
the  clear  serum,  which  has  separated  from  the  clot,  into  a  sterile 
test-tube  plugged  with  absorbent  cotton,  and  to  put  it  into  the 
ice  chest  away  from  the  light.     To  reassure  its  perfect  preser- 
vation one  may  add  to  it  some  phenol  in  such  proportion  that 
the  carbolic  is  present  to  the  extent  of  a  1/2  per  cent,  solution, 
e.g.,  to  9  c.cm.  of  serum  add  i  c.cm.  of  a  5  per  cent,  phenol 
solution.     The  latter  should  be  added  drop  by  drop  and  ag- 
itated, so  as  to  avoid  the  formation  of  precipitates.     Another 
method,  which  the  author  employs  almost  exclusively  for  the 
preservation  of  sera  containing  amboceptors,  consists  simply 
in  heating  the  sera  at  56°  C.  for  a  half  hour  and  then  placing 
them  into  the  ice  chest.     Inactivation  has  the  advantage  of 
stopping  molecular  changes  produced   by  ferment  actions  of 
fresh  serum.     Furthermore,  heating  acts  as  a  sterilizer  for  iso- 
lated air  germs  which  may  have  found  their  way  into  the  serum 
during  the  process  of  getting  it.     In  this  form,  a  serum  can  be 
kept  in  the  ice  box  for  several  weeks  without  any  material 
change.     Occasionally  one  finds   that  a  serum  will  undergo  Tube  used 
contamination  in  spite  of  inactivation,  so  that  if  a  serum  is  to  for  preser- 
be  preserved  for  several  months,  it  is  advisable  to  seal  it  in  a  vation  of 
test-tube.     For  this  purpose  a  brown  glass  tube  slightly  drawn 


out  at  its  upper  end  is  employed  (Fig.  8)  .  The  serum  is  placed 
into  this  sterilized  tube  and  then  the  latter  is  sealed  in  the  flame  at  its 
narrow  part.  Bacterial  and  organ  extracts  are  well  kept  in  this  way.  The 
best  method  of  preservation  consists  in  evaporating  the  serum  to  dryness 
in  a  vacuum  desiccator.  This  procedure  is  rather  complicated  and  can 
therefore  be  employed  only  in  institutions. 

A  vacuum  desiccator  with  beatable  plates  is  used.  The  serum  is  poured  in  very 
thin  layers  in  sterile  flat  dishes  and  allowed  to  dry  out  in  the  desiccator  at  a  tempera- 
ture of  30°  C.,  later  on  at  35°  C.  in  a  vacuum  of  3  cm.  mercury.  The  dried  serum 


i6 


LABORATORY  EQUIPMENT 


forms  a  yellowish-red  horny  mass  which  is  scraped  off  from  the  dish  and  ground  up  in 
a  mortar  into  a  yellowish  powder.  The  serum  powder  is  then  sealed  in  a  brown 
glass  tube. 

When  this  dried  serum  is  to  be  used,  the  tip  of  the  ampoule  is  broken  off,  and  several 
drops  of  isotonic  salt  solution  at  a  temperature  of  30°  C.  are  poured  in,  in  just  sufficient 
an  amount  to  moisten  the  wall  of  the  glass  tube.  By  rolling  the  tube  to  and  fro,  one 
finds  that  the  serum  powder  will  easily  stick  to  the  moistened  wall.  The  granules  are 
allowed  to  swell  up  and  after  they  have  done  so,  enough  isotonic  salt  solution  is  added 
to  make  up  the  original  volume. 

For  the  preservation  of  thermolabile  substances,  the  method  of  freezing  has  been 
suggested.  Morgenroth  has  devised  for  this  purpose  a  simple  and  handy  apparatus 
named  Frigo  which  can  be  obtained  from  Lautenschlager ,  Berlin.  Although  for  most 
tests  this  method  of  preservation  has  been  employed  with  success,  Neisser's  clinic 
reports  that  sera  preserved  in  the  Frigo  with  the  idea  of  retaining  their  complement 
did  not  give  as  accurate  complement  fixation  experiments  as  did  similar  fresh  sera. 

Friedberger  advises  the  addition  of  8  per  cent,  salt  solution  for  the  preservation  of 
the  complement.  When  the  serum  is  to  be  used  it  is  diluted  tenfold  with  distilled 
water,  so  that  a  10  per  cent,  dilution  of  complement  is  obtained.  By  the  addition  of 
the  salt,  the  resistance  against  harmful  effects  of  light,  room  and  body  temperature, 
and  chemical  substances  like  phenol  is  increased,  but  the  thermolability  of  the  com- 
plement remains  the  same.  Drying  a  serum  in  a  desiccator  is  not  to  be  advocated 
for  the  preservation  of  the  complement,  as  during  such  procedure  a  portion  of  the 
complement  is  lost.  Once  the  serum  is  in  its  dried  form,  however,  the  remaining 
complement  is  retained  and  in  addition,  has  become  resistant  against  high  heat. 


Filtration  of  Bacteria. 

It  is  important  in  many  serological  studies  to  be  able  to  separate  bac- 
teria from  their  fluid  media  or  suspension.  This  is  accomplished  either  by 
centrifugalization  or  filtration.  The  first  method  does  not  completely 


FIG.  9.— Pukal  filter. 


FIG.  10. — Filtration  through  a  Pukal  filter. 


free  the  fluid  of  its  bacteria,  but  if  this  is  desired  the  method  of  filtration  is 
essential.  In  this  connection,  however,  one  must  bear  in  mind  that  many 
albuminous,  or  albumen-like  substances,  few  colloids  and  even  some  toxins, 
do  not  pass  the  filters  and  are  therefore  held  back.  Bacterial  filtration  is 
simplified  by  preliminary  centrifugalization  or  passing  the  fluid  through 


FILTRATION   OF  BACTERIA  17 

filter-paper.  Different  porous  materials  have  been  used  for  bacterial 
filters,  of  which  especially  suitable  are  porcelain,  infusorial  earth  and  as- 
bestos. The  filtration  apparatus  consists  of  the  respective  filter  and  the 
receptacle  which  receives  the  filtrate.  Filtration  takes  place  by  differ- 
ences in  pressure,  where  either  the  fluid  is  forced  through  by  high  pressure 
or  sucked  through  by  a  vacuum  formed  in  the  receiving  vessel.  The 
following  are  some  of  the  filters  most  commonly  in  use. 

1.  CHAMBERLAIN'S  CYLINDER  FILTER,  F,  used  in  the  Pasteur  Institute  at  Paris. 
The  filter  cylinder  is  made  of  infusorial  earth  and  may  be  attached  to  any  water  outlet. 

2.  PUKAL  FILTER,  made  of  burnt  kaolin,  is  used  especially  for  the  filtration  of  large 
quantities  of  fluid.     The  filter  b  is  placed  into  the  beaker  e  containing  the  toxin  and 
bacterial  fluid.     The  filter  is  then  closed  by  a  rubber  stopper,  perforated  by  a  central 
opening  through  which  runs  a  glass  tube  bent  at  right  angles,  and  this  in  turn  is  con- 
nected with  either  an  air  or  water  pump  for  producing  a  vacuum  inside  of  the  filter. 
Between  the  filter  and  vacuum  pump  can  be  interposed  a  sterile  jar  a  (Figs.  9  and  10). 


FIG.  ii.— Reichel  filter. 


FIG.  12. — Lilliputian  filter. 


3.  THE  REICHEL  FILTER  (Fig.  1 1)  consists  of  a  glass  receiver  A,  having  a  side  neck  c 
and  at' the  bottom  a  tube-like  outlet  d.     A  porcelain  filter  B  fits  into  the  glass  jar  and 
rests  upon  the  margin  of  the  flask  by  means  of  a  broad  collar.     The  point  of  junction 
is  made  air  tight  by  means  of  a  rubber  cap  with  a  central  opening,  through  which  the 
cylinder  can  be  filled.     When  in  use  d  is  shut  off  by  a  rubber  tube  with  a  pinch  cock 
and  c  in  which  lodges  a  small  piece  of  cotton  is  connected  with  a  water  pump  that  is 
instrumental  in  bringing  about  a  vacuum.     The  function  of  d  is  to  allow  the  removal  of 
samples  of  the  filtrate  and  finally  to  obtain  the  entire  filtrate. 

4.  THE  LILLIPUTIAN  FILTER,  candle-like  in  shape,  and  made  of  infusorial  earth,  is 
employed  for  the  filtration  of  very  small  quantities.     The  filter  is  cemented  upon  a 
metal  tube  which  is  screwed,  so  that  it  is  air  tight,  into  a  well-fitting  glass  cylinder  open 
at  the  top.     The  tube  is  passed  through  a  rubber  cork  which  tightly  closes  an  exhaust 


i8 


LABORATORY  EQUIPMENT 


flask.  The  fluid  to  be  filtered  is  placed  into  the  glass  cylinder  and  sucked  through  into 
the  flask  by  means  of  a  vacuum  produced  here.  For  the  purpose  of  collecting  very 
small  quantities  a  test-tube  may  be  placed  into  the  exhaust  flask  (Fig.  12). 


Preparation  of  Dilutions  and  Measurement  of  Small  Amounts  of 

Bacteria. 

All  serological  methods  are  to  be  considered  on  quantitative  bases.  In 
serum  diagnosis  as  well  as  in  the  therapy,  the  amount  of  the  serum- employed 
is  the  deciding  factor.  Similarly,  the  number  or  amount  of  bacteria  required 
either  for  the  purposes  of  immunization  or  serological  reactions  is  of  ex- 
treme importance. 

One  cubic  centimeter  is  the  unit  of  measure  for  serum  and  all  fluid  mate- 
rial (Bouillon  cultures,  exudates,  etc.).  //  small  quantities  are  required,  it 
is  best  to  dilute  the  fluid  with  0.85  per  cent,  saline  solution.  The  exact  prepa- 
ration of  dilutions  is  one  of  the  most  essential  technical  procedures  in  serum 
diagnosis.  Some  general  rules  may  be  of  help. 

1.  Never  should  amounts  less  than  o.i  c.cm.  be  measured  out  directly. 
For  beginners  even  o.i  is  best  measured  in  the  form  of  a  dilution,  as  errors 
are  apt  to  occur  very  easily. 

2.  The  decimal  system  should  be  adhered  to  as  much  as  possible. 

3.  The  dilution  should  be  made  just  before  it  is  to  be  used,  inasmuch 
as  many  substances  retain  their  activity  best,  or  only,  in  concentrated 
form. 

The  following  is  an  example  of  correct  forms  of  dilutions: 


Toxin 

Dilution  of  toxin, 

Dilution  of  toxin, 

Dilution  of  toxin, 

i  no 

i  :ioo 

i  :  1000 

o.i  c.cm.  = 

i      c.cm. 

0.05  c.cm.  = 

0.5  c.cm. 

o.o  i  c.cm.  = 

o.  i  c.cm. 

=  i      c.cm. 

0.005  c.cm.  = 

0.5  c.cm. 

o.ooi  c.cm.  = 

o.i  c.cm. 

=  1.0  c.cm. 

0.0005  c.cm.  = 

0.5  c.cm. 

The  stock  dilution  of  i  :  10  is  made  by  measuring  off  i  c.cm.  of  toxin  and  adding 
9  c.cm.  of  0.85  per  cent,  of  saline. 

The  dilution  i :  100  can  be  made  by  taking  i  c.cm.  of  toxin  and  adding  99  c.cm.  of 
saline.  It  is  more  practicable,  however,  to  take  i  c.cm.  of  the  i  :  10  stock  dilution 
and  add  9  c.cm.  of  saline.  If  the  dilution  i :  ib  is  not  present  and  only  a  small  amount 
of  the  dilution  i  :  100  is  desired,  the  latter  is  made  by  taking  o.i  toxin  :  10.0  NaCl  sol. 
Similarly  1:1000  =  0.1:100=1  c.cm.  of  the  dilution  (i  :  10)  :ioo=i  c.cm.  of  the 
dilution  (i  :ioo)  :io.o. 


PREPARATION   OF  DILUTIONS 
The  following  table  shows  the  details  of  various  dilutions: 


Dilution  i  :  10 

Dilution  i  :  100 

Dilution  i  :  1000 

Dilution  i  :  10,000 

T  c.cm.  +9  c.cm.  NaCl 

i  c.cm.  +99  c.cm.  Na- 

i c.cm.  +999  NaCl  = 

o.  i  c.cm.+999.9  c.cm. 

sol.  =  o.i  c.cm.  +0.9 

Cl  sol.  =  o.  i  c.cm.+ 

o.  i  c.cm.+99.9  c.cm. 

NaCl  sol.  = 

c.cm.  NaCl  solution. 

9  .  9  c.cm.  NaCl  solu- 

NaCl= 

i  c.cm.  of  dilution 

=  0.2  c.cm.  +1.8  c. 

tion. 

o.  2  c.cm.  +  199  .  8  c.cm. 

i  :  1000 

cm.  NaCl  solution. 

=  0.2  c.cm.+  i9.8  c.cm. 

NaCl  sol. 

+9  c.cm.  of  NaCl  sol. 

=0.3  c.cm.  +  2.7  c.cm. 

NaCl  solution. 

=  2  c.cm.+  i998  c.cm. 

=0.1  c.cm.  of  dilu- 

NaCl solution. 

=  0.3  c.cm.  +29.  7  c*.cm. 

NaCl  sol. 

tion  i  :  100 

NaCl  sol. 

=  i  c.cm.  of  dilution 

+9.9  c.cm.  NaCl  sol. 

=  3  c.cm.  +27  c.cm. 

=  i  c.cm.  of  dil.  i  :  10 

i  :  100+9  c.cm.  NaCl 

=  i  c.cm.  dil.  i  :  100 

NaCl  sol. 

+9  c.cm.  of  NaCl.  sol. 

sol.  =0.1  c.cm.  dil. 

+99  c.cm.  NaCl.  sol. 

=  10  c.cm.+go  c.cm. 

=  2  c.cm.  of  dil.  i  :  10 

i  :  10 

=  0.1  c.cm.  dil.  i  :  10. 

NaCl  sol. 

+  18  c.cm.  NaCl  sol., 

+9.9  c.cm.  NaCl  sol. 

+99  .  9  c.cm.  NaCl.  sol. 

etc. 

=  o.  i  c.cm.  of  dil. 

i  :  100+0.9  c.cm. 

' 

NaCl  solution. 

In  preparing  these  dilutions,  it  is  best  to  measure  off  the  small  quantities  o.i- 
i  c.cm.  with  a  pipette;  allow  this  to  run  into  a  well-graduated  measuring  glass  and  add 
enough  saline  to  make  the  required  dilutions.  For  example,  if  30  c.cm.  of  a  dilution 
i :  100  is  desired,  0.3  c.cm.  should  be  measured  off  with  a  pipette  and  aUowed  to  flow 
into  a  50  or  100  c.cm.  graduated  cylinder  and  saline  solution  added  up  to  30.0  c.cm. 

It  should  always  be  one's  aim  to  get  along  with  small  quantities  of  the  substance 
to  be  diluted.  If,  for  example,  8  to  10  c.cm.  of  a  toxin  dilution  i  :  100  are  required  o.i 
c.cm.  of  toxin  +9.9  c.cm.  of  saline  should  be  taken  and  not  i  c.cm.  of  toxin  and  99  c.cm. 
of  NaCl  sol. 

Before  making  any  dilution  one  should  always  calculate  the  total  amount  of  sub- 
stance required;  as  for  example  in  the  following  experiment: 

1.  Animal  o.i      c.cm.  Toxin  subcutaneously 

2.  Animal  0.05    c.cm.  Toxin  subcutaneously 

3.  Animal  o.oi    c.cm.  Toxin  subcutaneously 

4.  Animal  o.ooi  c.cm.  Toxin  subcutaneously 

Here,  the  total  quantity  of  toxin  necessary  is  found,  by  adding,  to  be  0.161  c.cm. 
This  represents  the  minimum  amount.  It  is  always  advisable  to  make  an  allowance 
for  some  loss  and  at  the  same  time  bring  up  the  amount  to  a  round  or  even  number. 
0.2  c.cm.  of  toxin  would  fulfill  all  these  requirements.  This  amount  is  measured  off 
by  a  pipette,  placed  into  a  graduated  cylinder  and  saline  added  up  to  2.0  c.cm.,  making 
a  dilution  of  i :  10.  Then  0.2  c.cm.  of  this  dilution  (i  :  10)  is  taken,  placed  into  another 
graduate,  and  again  diluted  with  saline  up  to  2.0  c.cm.  thus  making  a  dilution  of 
1:100.  The  above  problem  therefore  of  injecting  the  various  animals,  ran  be  com- 
pleted as  follows: 

1.  Animal  receives  i     c.cm.  of  dilution  i :  10 

2.  Animal  receives  0.5  c.cm.  of  dilution  i  :  10 

3.  Animal  receives  i     c.cm.  of  dilution  i  :  100 

4.  Animal  receives  o.i  c.cm.  of  dilution  i  :  100 


20  LABORATORY  EQUIPMENT 

The  unit  for  measuring  the  amount  of  bacteria  grown  upon  a  solid 
medium  is  represented  by  a  standard  sized  loop.  This  platinum  loop  takes 
up  about  2  mg.  of  bacterial  substance.  It  is  prepared  as  explained  in  Fig. 
2.  If  smaller  amounts  of  bacteria  are  used,  dilutions  must  be  made. 

For  instance.  1/4  of  a  loopful  of  bacteria  is  desired;  i  loopful  is  suspended  in  i  c.cm. 
of  saline  and  3  c.cm.  of  saline  added.  As  a  result,  i  c.cm.  of  this  emulsion  contains  1/4 
of  a  loopful  of  bacteria.  If  1/16  of  a  loopful  is  necessary  i  c.cm.  of  the  above  dilution 
is  added  to  3  c.cm.  of  saline,  thus  making  i  c.cm.  of  this  last  mixture  contain  1/16 
of  a  loopful  of  bacteria. 


CHAPTER  III. 

ACTIVE  IMMUNIZATION. 

IMMUNIZATION  WITH  LIVING  AND  DEAD  VIRUS. 

Active  immunization  depends  upon  the  principle,  that  an  organism  in 
overcoming  a  slight  infection,  either  naturally  or  artificially 

ncip  e  acquired,  develops  enough  protective  bodies  to  withstand  a 
of  Active       .     .,  .      ,   .    ,    ^. 

Immun-       similar,  severer,  natural,  or  acquired  infection.     Moreover, 

ization.  ^  serves  primarily  the  purpose  of  prophylaxis.  In  labora- 
tories, active  immunization  of  animals  is  also  frequently  under- 
taken with  the  view  of  obtaining  sera  for  diagnostic  and  therapeutic 
purposes. 

In  the  manufacture  of  large  quantities  of  serum,  the  horse  is  the  animal 
used  almost  exclusively.  Occasionally  cows,  sheep,  donkeys  or  mules' 
are  selected.  In  small  laboratories  usually  rabbits,  guinea-pigs,  white 
mice,  rats,  and  only  occasionally  goats  or  sheep  are  employed. 

The  process  of  immunization  evokes  a  marked  disturbance  in  the  gen- 
eral health  of  the  animals.  For  this  reason  they  must  be  well  kept  in 
warm  places,  and  well  fed.  As  far  as  their  power  of  producing  antibodies 
is  concerned,  there  are  individual  differences  even  among  the  same  species 
of  animals;  thus  if  five  horses  are  immunized  against  diphtheria,  some  will 
give  much  better  curative  sera  than  the  others.  In  general,  the  younger 
animals  are  preferable. 

Any  substance  which,  when  injected  into  an  organism,  can  stimu- 
late the  production  or  formation  of  antibodies,  has  been  conveniently 
termed  "  antigen."  After  the  injection  of  such  an  antigen,  special  notice 
should  be  taken  of  the  animal  in  reference  to  temperature,  weight,  the 
excitation  of  diarrhea  or  the  occurrence  of  abscesses,  infiltrates,  edema 
or  paralysis. 

If  an  animal  dies,  a  careful  postmortem,  and  if  possible,  a  bacteriolog- 
ical examination  should  be  made.  It  should  be  the  aim  to  ascertain  if  death 
was  induced  by  the  inoculated  antigen,  by  contamination  or  secondary 
infection.  One  should  always  keep  in  mind  the  possibility  of  some  of  the 
animal  epidemic  diseases. 

Epidemic  diseases  occurring  in  rabbits  are: 

Animal      i.  RABBIT  SEPSIS. — Presents  itself  in  the  form  of  bronchopneumonia  and 
Infections,   marked  nasal  catarrh.     It  is  very  infectious.     Sick  animals  should  at 
once  be  isolated  or  killed  and  their  cages  thoroughly  disinfected. 

21 


22  ACTIVE  IMMUNIZATION 

2.   COCCIDIOSIS  gives  changes  in  the  liver  due  to  the  settling  of  the  coccidi  ova 
forms.     The  parasites  are  present  in  the  pus  and  are  easily  recognized  microscopically. 
Following  labor,  guinea-pigs  are  very  susceptible  to  sepsis. 
IN  RATS,  trypanosomiasis  is  of  frequent  existence,  but  is  not  pathogenic. 

The  antigens  are  injected  either  subcutaneously ,  intraperito- 
The  Tech-   neally  or  intravenously.     Only  on    exceptional  occasions  is 
nique  of     another  entrance  path  chosen. 

Active  Im-  As  regards  the  amount  to  be  injected,  one  cannot  very  well 
mumzation.  g|ve  generai  rules.     It  is  important  to  prevent  severe  re- 
actions, although  the  question  is  still  a  disputed  one,  whether 
marked  reactions  tend  to  produce  a  better  immunity.     It  is  certain,  how- 
ever, that  inoculations  of  antigens  in  such  minute  doses  as  to  apparently 
give   no    reaction,    can  still  lead  to  immunity  and  the  production  of 
antibodies. 

Occasionally  a  single  injection  suffices  for  immunization.  Repeated 
inoculations  are  usually  necessary,  especially  so  when  a  "highly  valent" 
serum  is  desired,  i.e.,  one  containing  a  great  number  of  antibodies  or  having 
high  protective  properties. 

When  repeated  inoculations  are  undertaken,  there  are  various  methods 
of  procedure. 

i a.  A  small  dose  of  antigen  is  injected.  If  a  reaction  sets  in,  one  waits 
until  this  reaction  has  entirely  subsided,  then  (not  before  the  fifth  day) 
the  second  injection — a  somewhat  larger  dose — is  given.  After  an  interval 
of  5  to  8  days,  a  third  injection  of  a  still  higher  dosage  is  administered, 
and  so  on,  again. 

ib.  The  intervals  are  the  same,  but  the  amounts  of  antigen  remain  the 
same  at  each  injection. 

Both  of  these  methods  give  excellent  results  and  therefore  are  most 
frequently  used. 

2.  For  several  successive  days,  a  small  or  medium  dose  of  antigen  is 
injected.     Each  injection  produces  only  a  slight  reaction. 

This  last  scheme  according  to  Fornet  is  especially  suitable  for  obtain- 
ing precipitation  sera.  As  is  evident,  it  has  the  advantage  of  gaining  the 
immunity  rapidly. 

3.  Inoculations  are  given  at  very  long  intervals  (intermissions  of  four 
weeks  or  more).     This  method  produces  good  sera,  but  has  the  disad- 
vantage of  requiring  too  long  a  time. 

The  methods  of  active  immunization  can  also  be  divided  according  to 
the  nature  of  the  antigen. 

1.  Immunization  with  a  living  virus, 

2.  Immunization  with  a  dead  virus, 

3.  Immunization  with  bacterial  extracts, 

4.  Immunization  with  bacterial  toxins. 


CLASSIFICATION   OF  BACTERIA 


I.  Immunization  with  a  Living  Virus. 


23 


This  method  of  immunization  simulates  most  closely  the  immunity 
attained  spontaneously  in  overcoming  an  infection.  This  immunity 
is  very  strong  and  lasts  for  a  long  period  of  time,  but  it  is  attained  with 
difficulty;  frequently  the  dose  of  virus  injected  causes  serious  symptoms  of 
infection.  Various  procedures  have  therefore  been  advocated  so  to  di- 
minish the  toxicity  of  the  immunizing  agent  that  only  immunization 
effects,  and  no  toxic  symptoms  be  obtained.  This  was  attempted  either 
by  the  reduction  of  the  number  of  organisms  employed,  so  that  very 
minute  doses  were  inoculated,  or  by  the  diminution  of  the  infectious 
nature  of  these  bacteria  (virulence  so  called). 

The  first  method,  however,  was  not  found  applicable  to  all  cases.  The 
infectious  nature  of  the  different  bacteria  varies  markedly.  The  same 
bacterium  reacts  differently  with  different  animals.  While  some  animals 
possess  a  natural  immunity  against  certain  bacteria,  others  exhibit  a 
distinct  susceptibility  to  the  same  micro-organisms.  The  conceptions 
therefore  of  pathogenicity  and  virulence  are  purely  of  a  relative  nature.  '  In 
talking  of  the  pathogenicity  of  bacteria,  one  should  always  mention  the  class 
of  animal  for  which  these  bacteria  are  pathogenic. 

Bail  has  used  this  principle  of  pathogenicity  in  classifying  bacteria.     He 
Bail's  Classi-  mentions  the  following  three  classes: 
fication  of    a.  Saprophytes. 
Bacteria,     b.  Half  or  partial  parasites. 
c.  Whole  or  pure  parasites. 

To  the  class  of  saprophytes  belong  all  those  bacteria  which  when  injected  even  in 
larger  doses  do  not  produce  any  characteristic  disease;  these  are  also  known  as  apatho- 
genic — e.g.,  hen  cholera  bacilli  for  human  beings. 

Classed  as  half  parasites  are  those  bacteria,  according  to  Bail,  the  infectious  nature 
of  which  depends  upon  the  quantity  of  bacteria  injected.  While  the  injection  of  a 
rabbit  with  i/iooo  of  a  loopful  of  a  typhoid  culture  will  produce  no  evidences  of  disease, 
one-tenth  of  a  loopful  will  result  in  slight  increase  in  temperature,  loss  of  appetite,  and 
eventually  a  local  redness  at  the  site  of  the  injection.  One  loopful  may  bring  about 
the  death  of  the  animal.  The  manifestations  are  dependent  entirely  upon  the  number 
of  bacteria  injected.  The  smaller  the  number,  the  milder  the  symptoms,  until  one 
reaches  the  stage  below  which  no  disturbances  at  all  are  visible. 

Pure  parasites  are  those  which  have  no  sublethal  dose.  Even  the  smallest  amount, 
when  injected,  will  produce  death.  As  examples,  the  tubercle  bacillus  for  guinea-pigs, 
and  bacilli  belonging  to  the  group  of  Hemorrhagic  Septicemia  for  rabbits.  Of  the  last 
mentioned  1/10,000,000,000  of  a  loopful  of  some  cultures  kills  a  rabbit  within  twenty- 
four  hours  with  the  symptoms  of  a  septicemia;  in  other  words,  the  injection  of  i  c.cm. 
of  a  dilution  of , one  loopful  of  culture  in  ten  million  liters  of  water  suffices  to  kill  the 
rabbit.  Furthermore,  the  bacteria  increase  so  greatly  in  the  body  of  the  rabbit  that 
they  can  be  demonstrated  in  every  drop  of  blood  and  in  all  organs  and  body  fluids. 

The  same  organism  is  a  saprophyte  for  the  human  being  and  a  half  parasite  for  the 
guinea-pig  if  injected  subcutaneously  and  a  complete  parasite  by  intraperitoneal  injec- 


24  ACTIVE  IMMUNIZATION 

tion.  The  conceptions  therefore  of  complete  or  partial  parasite  as  well  as  of  saprophyte 
are  only  relative  and  are  dependent  upon  the  bacteria,  the  animal  species,  and  the  mode  of 
infection. 

It  is  now  clear  that  immunization  with  living  bacteria  can 
Example  of  onty  ^e  undertaken  if  the  latter  belong  to  the  class  of  half- 
Active  Im-  parasites.  Pure  parasites  are  excluded  from  this  method, 
munization  As  an  example  of  such  procedure  can  be  given  the  immu- 
with  Living  Cation  of  a  guinea-pig  by  intraperitoneal  injections  with 
Bacilli.  }iving  typhoid  bacilli.  Preliminary  to  this,  the  virulence 

of  the  typhoid  culture  must  be  ascertained. 

a.  Preliminary  test  to  titrate  the  virulence  of  the  typhoid  culture. 

1.  Guinea-pig  i./I.  1909  1/20  loopful  of  typhoid  culture  intraperitoneal. 

2. /I.  active. 

8./I.  alive. 

2.  Guinea-pig  i./I.  1909  i/io  loopful  of  typhoid  culture  intraperitoneal. 

2. /I.  active. 

8./I.  alive. 

3.  Guinea-pig  i./I.  1909  1/8  loopful  of  typhoid  culture  intraperitoneal. 

2./I.  slightly  sick,  does  not  eat. 

3./I.  active. 

8./I.          alive. 

4.  Guinea-pig  i./I.  1909  1/6  loopful  of  typhoid  culture  intraperitoneal. 

2./I.  sick,  does  not  eat,  hair  raised. 

3./I.  still  sick. 

4./I.  more  active. 

8./I.  alive. 

5.  Guinea-pig  i./I.  1909  1/5  loopful  of  typhoid  culture  intraperitoneal. 

2. /I.          sick,  does  not  eat,  hair  raised. 

3 ./I.  very  weak,  when  placed  on  side  remains  so. 

4./L  f 

6.  Guinea-pig  i./I.  1909  1/4  loopful  of  typhoid  culture  intraperitoneal. 

,./!.  f 

From  this  experiment  it  becomes  evident  that  the  lethal  dose  of  this  particular 
strain  of  typhoid  culture  is  1/4  to  1/5  of  a  loopful  for  guinea-pigs  by  intraperitoneal 
injection.  Immunization  therefore  must  be  started  with  a  smaller  dose — e.g.,  i/io 
of  a  loopful. 

b.  Immunization. 

1.  Guinea-pig    8./I.  1909  i/io  loopful  of  typhoid  culture  intraperitoneal. 

i6./I.  1/8  loopful  of  typhoid  culture  intraperitoneal.     . 

2 2. /I.  1/4  loopful  of  typhoid  culture  intraperitoneal. 

Animal  remains  active  and  healthy. 
3O./I.  i  loopful  of  typhoid  culture  intraperitoneal. 

5-/II.  2  loopfuls  of  typhoid  culture  intraperitoneal. 

Animal  remains  active  and  healthy. 

2.  Guinea-pig    8. /I.  1909  i/io  loopful  of  typhoid  culture  intraperitoneal. 

i6./I.  1/4  loopful  of  typhoid  culture  intraperitoneal. 

Animal  remains  active  and  healthy. 


VACCINATION   AGAINST    SMALL-POX  25 

3.  Guinea-pig   8./I.  1909  i/io  loopful  of  typhoid  culture  intraperitoneal. 
i6./I.  2  loopfuls  of  typhoid  culture  intraperitoneal. 

I7./I.  animal  is  sick  and  does  not  eat. 

i8./I.  animal  is  very  weak. 

I9-/I.  t 

Control  animals  always  die  within  twenty-four  hours,  as  in  previous  experiment,  on 
injection  of  1/4  of  a  loopful. 

From  experiment  with  guinea-pig  i,  it  can  be  learned,  that  by  gradual  increase  of 
the  immunizing  dose,  a  state  of  immunity  is  reached  which  can  overcome  an  infection 
produced  by  a  high  multiple  of  the  dosis  letalis. 

Experiments  2  and  3  prove  that  even  a  single  preliminary  injection  suffices  to  pre- 
vent the  death  of  an  animal  upon  subsequent  receipt  of  the  lethal  dose  of  the  same 
bacteria;  but  this  single  inoculation  is  not  sufficient  to  protect  the  organism  against  a 
very  severe  future  infection.  The  attained  immunity  is  therefore  only  relative,  not 
absolute. 

Analogously  it  is  possible  to  immunize  by  subcutaneous  and  intra- 
venous injections.  The  latter  method  is  usually  the  one  of  choice  when 
half  parasites  are  employed,  as  the  highest  and  quickest  grade  of  immunity 
is  thus  reached.  It  carries  with  it,  however,  the  greatest  danger,  and  fre- 
quently results  in  death  of  the  animal. 

The  method  of  immunization  with  small  doses  of  living,  fully  virulent 
bacteria,  has  thus  far  been  made  use  of  only  in  animals.  In  man  this 
experience  has  not  been  carried  into  effect.  It  is  j eared  that  the  bacteria  may 
increase  very  rapidly  and  give  rise  to  severe  disturbances.  The  method  has 
therefore  been  altered  and  instead  of  using  virulent  material  for  immu- 
nization, only  a  weakly  infectious  or  attenuated  virus  is  employed. 


Vaccination  against  Small-pox. 

This    is    the    best    known    example    of    active    prophylactic 
immunization.     To  Jenner  belongs  the  credit  of  having  been 
the  first   one  to   apply  this  principle.     Vaccination   against 
small-pox  consists  in  inoculation  of   an  attenuated  form  of 
small-pox  germs,  the  diminution  in  virulence  being  brought  about  by  pass- 
age through  the  body  of  a  calf,  a  less  susceptible  animal  than  man.     The 
vesicles  formed  on  the  vaccinated  person  contain  these  attenuated  germs. 
This  lymph  can  be  used  for  the  inoculation  of  other  individuals,  as  the 
germs  do  not  regain  their  virulence  by  repassage  through  man. 

Inasmuch  as  it  is  not  within  the  scope  of  this  book  to  go  into  the  details 
of  the  preparation  of  the  lymph  or  the  technique  of  vaccination,  a  brief 
survey  of  the  benefits  of  vaccination  will  amply  suffice  and  this  may  be 
seen  from  the  table  hereunto  appended. 

The  mortality  from  small-pox  per  100,000  population  was  in  the  years 


26 


ACTIVE  IMMUNIZATION 


in  Prussia  and  Bavaria 

in  Austria 

in  Belgium 

in  England 

in  Sweden  . . 


1882-1896 

0.7 

38.6 

18.2 

2.9 


1862-1876 

Si.6 

75-2 

79-5 

25.3 

26.9  0.5 

This  method  of  immunizing  against  a  virulent  virus  by  inoculating  with  an 
attenuated  form  of  the  same,  is  known  as  Jennerization.  Pasteur  recog- 
nized that  this  method  had  general  application  and  similarly  used  attenuated 
but  still  living  cultures,  "vaccins"  so  called,  to  immunize  against  hen  cholera, 
swine  plague,  and  anthrax.  The  same  principle  underlies  Pasteur's  anti- 
rabic vaccination. 

Antirabic  Vaccination. 

In  all  civilized  countries  there  exist  special  institutions,  either  directly 
under  the  city  control  or  appointed  by  the  city,  where  the  Pasteur  treat- 
ment for  rabies  is  conducted.  It  is  the  duty  of  the  general  practitioner, 
on  getting  a  suspicious  case  of  rabies,  to  advise  his  patient  to  undergo  this 
special  therapy  and  to  send  the  rabid  animal,  its  head  or  brain  preserved 
in  glycerin,  to  the  institute  as  soon  as  possible  for  the  purpose  of  ascer- 
taining the  presence  of  rabies.  Up  to  very  recently  the  actual  cause 
of  hydrophobia  was  unknown.  Negri  had  described  parasites,  known 
as  Negri  bodies,  in  the  large  nerve  cells  of  the  cerebral  cortex, 
cerebellum,  etc.1  In  man,  infection  usually  occurs  as  a  con- 
Treatment  se°iuence  °f  the  saliva  of  rabid  animals  (dog,  cat,  wolf,  skunk) 
gaining  entrance  to  wounds  from  bites  or  scratches.  Roux 
and  Nocard  found  that  the  saliva  of  experimentally  infected  animals  is 
already  infectious  2-3  days  before  the  first  symptoms  appear.  Thus, 
rabies  may  be  transmitted  to  an  individual  by  an  animal  apparently 
healthy  at  the  time.  Remlinger  has  formulated  the  following  very  instruc- 
tive outline  referring  to  the  indications  for  antirabic  treatment. 

1.  has  died  within  10  days  after  the  biting, 

2.  has  been  killed  within  10  days  after  the  biting, 

3.  has  disappeared  within  10  days  after  the  biting, 

4.  is  unknown  to  the  individual  bitten, 

(a)  becomes  ill  with  rabies, 

(b)  dies  with  suspicious  symp- 
toms of  rabies  or  of  another 
disease, 


If  the 
biting 
animal 


5.  has  remained  alive 
and  under  observa- 
tion for  10  days, 


antirabic 

treatment 

is  indicated. 


antirabic 

treatment 

is  indicated. 


(c)  becomes   ill   but   does 
die  within  10  days, 


not 


(d)  remains   well   both    during 

and  after  this  period, 
1  Noguchi  has  now  succeeded  in  artificially  cultivating  the  rabic  virus. 


further 
observation, 
treatment  if 
animal  dies. 

no 

treatment. 
By  inoculating 


cultures  containing  these  granular,  pleomorphic  or  nucleated  bodies,  he  has  reproduced  rabies 
in  dogs,  rabbits  and  guinea-pigs. 


PASTEUR   TREATMENT   FOR   RABIES  27 

Pasteur  found  that  rabies  can  be  transmitted  to  dogs  by  injecting  them 
subdurally  with  the  brain  substance  of  rabid  animals.  This  ordinary  virus 
substance  is  known  as  Street  Virus. 

The  incubation  period  of  rabies  is  very  long.  It  varies  from  about  three 
weeks  to  [possibly]  some  years.  By  passing  the  virus  through  monkeys, 
the  incubation  period  is  considerably  increased.  After  the  successive 
passage  through  five  or  six  animals,  the  virus  becomes  so  weakened  that 
infection  is  almost  impossible.  Reversely,  increase  of  the  virulence  may 
be  affected  by  passing  the  virus  through  a  successive  number  of  rabbits, 
as  these  are  very  sensitive  to  the  disease.  After  passage  through  a  large 
number  of  such  animals,  the  incubation  period  is  gradually  shortened  from 
about  three  weeks  or  a  little  less  to  a  constant  period  of  six  or  seven  days. 
Further  diminution  in  the  period  of  incubation  was  impossible  and  there- 
fore Pasteur  called  this  "  Virus  fixe."  His  first  experiments  in  immuniza- 
tion were  made  by  passing  the  weakened  monkey  virus  through  rabbits 
and  then  treating  dogs  with  the  spinal  cords  of  the  latter. 

Later  on,  Pasteur  discovered  that  instead  of  passing  the  virus  through 
monkeys,  he  could  diminish  its  virulence  by  drying  the  spinal  cords  derived 
from  rabid  animals,  for  varying  periods  of  time.  In  this  way  he  could 
prepare  an  entire  series  of  graduated  strengths.  The  material  used  for 
this  drying  was  not  the  street  virus,  but  that  obtained  by  successive  pas- 
sage through  rabbits  or  "virus  fixe"  which  possessed  very  constant  im- 
munizing and  infectious  properties.  By  drying  the  "virus  fixe"  over 
caustic  potash  at  a  temperature  of  23°  to  25°  C.  for  five  days,  its  regular 
incubation  period  of  7  days  was  very  much  prolonged.  Increase  in  the 
length  of  drying  caused  the  entire  loss  of  virulence. 


Pasteur  immunized  dogs  as  follows:  He  began  with  the  injection  of  a  virulent 
spinal  cord  which  had  been  dried  for  thirteen  days  and  every  following  day  injected 
subcutaneously  some  fresher  spinal  cord,  i.e.  (dried  for  a  lesser  period  of  time),  until 
finally  he  used  virus  dried  only  for  one  day.  The  animals  thus  treated  were  immune 
against  the  bites  of  rabid  dogs  as  well  as  subdural,  subcutaneous,  and  intravenous 
infection  with  "virus  fixe  "  and  street  virus.  This  procedure  was  strongly  recom- 
mended by  Pasteur,  who  brilliantly  contributed  the  observation,  that  if  an  animal 
was'  infected  but  did  not  as  yet  show  symptoms,  these  could  be  prevented  by  a  similar 
modus  operandi,  as  above  mentioned. 


In  man,  the  inoculation  is  carried  out  on  the  same  principle.  The 
fact  that  the  incubation  period  of  hydrophobia  is  very  long,  makes  the 
prophylactic  inoculations  of  greater  service.  Only  rarely  is  this  period  less 
than  six  weeks,  usually  considerably  longer — up  to  584  days,  entirely 
dependent  upon  the  virulence  of  the  virus  and  the  point  of  infection. 


28 


ACTIVE  IMMUNIZATION 


Technique  of  Antirabic  Vaccination  in  Man. 

The  actual  vaccine  consists  of  i  c.cm.  (2-3  mm.  length)  of  the  substance 
of  the  spinal  cord  of  a  rabbit  which  has  been  killed  by  inoculation  with  the 
fixed  virus,  rubbed  up  into  a  fine  emulsion  with  5  c.cm.  of  sterile  0.85  NaCl 
solution.  About  i  to  3  c.cm.  of  the  resulting  fluid  are  injected  subcutane- 
ously  into  the  skin  of  the  abdomen.  A  cord  dried  for  fourteen  days  is  used 
for  the  first  injection,  emulsions  of  less  attenuated  virus  are  used  on  suc- 
ceeding occasions  until  finally  a  portion  of  a  spinal  cord  dried  for  only  three 
or  four  days  is  employed.  Pasteur's  schemes  of  the  actual  doses  can  thus 
be  drawn  up. 

a.  For  infections  at  points  distant  from  the  central  nervous  system  (mild  infections). 


Day  of  injection 

i 

2 

c; 

6 

7" 

8 

IO 

II 

12 

1-2 

14 

1C 

14 

12 

10 

8 

6 

5 

5 

4 

3 

5 

5 

4 

4 

3 

3 

Number  of  days  cord  was  dried  .  .  . 

+ 

+ 

+ 

+ 

+ 

13 

II 

9 

7 

6 

Amount  injected  in  cubic  centi- 

3-0 

3-0 

3-0 

3-0 

2.0 

I.O 

I.O 

I.O 

I.O 

2.O 

2.O 

2.O 

2.0 

2.0 

2.O 

meters. 

b.  For  head  wounds  (severer  infections). 


Day  of  injection. 

i 

2 

3 

4 

5 

6 

7 

8 

Number  of  days 
cord  was  dried. 

14            12 

10          8 

6+6 

5 

5 

4 

3 

4 

13            II 

In  A.  M.  In  p.  M. 

9           7 
In  A.  M.  In  p.  M. 

Amount  injected 
in  cubic  centi- 
meters. 

3-0    3-0 

A.  M.  P.  M. 

3-0     3-0 

A.  M.      P.  M. 

2  c.cm.     2  c.cm. 

A.  M.         P.  M. 

2. 

2. 

2. 

i. 

2. 

Day  of  injection  

TO 

TT 

14 

T  f 

1  6 

18 

TO 

Number  of  days  cord 

5 

5 

4 

4 

3 

3 

5 

4 

3 

5 

4 

was  dried. 

Amount  injected  

2. 

2. 

2. 

2. 

2. 

2. 

2. 

2. 

2. 

2. 

2. 

TECHNIQUE    OF   ANTIRABIC  VACCINATION 


29 


The  drawback  to  this  classical  method  of  Pasteur  consists  in  using  the 
virulent  material  rather  late  in  the  course  of  the  inoculations.  A  more 
energetic  treatment  has  therefore  been  advised.  There  is  no  added 
danger  in  doing  this  because  the  virus  fixe  in  contrast  to  the  street  virus  is  not 
at  all  or  only  slightly  infectious  for  man. 

Hogyes  in  Buda  Pesth  uses  the  virus  fixe  right  from  the  start.  He  be- 
gins with  marked  dilutions  (1/10,000)  and  gradually  increases  them  to 
i/ioo.  The  theory  underlying  this  procedure  is,  that  the  usual  method  of 
attenuation  by  drying  alters  the  quantity  of  the  virus  but  not  its  quality; 
hence  the  same  result  may  be  obtained  by  simple  dilution. 

Ferran  successfully  employs  the  virulent  virus  in  large  doses  right 
from  the  onset  of  the  treatment.  Especially  in  very  severe  infections,  as 
in  bites  from  wolves,  is  this  procedure  justifiable. 

The  exact  arrangement  of  doses  varies  a  little  at  different  institutions. 
In  Berlin,  it  is  considered  that  the  virulence  of  the  dried  cord  is  lost  on 
about  the  eighth  day  instead  of  the  fourteenth.  Hence  in  the  hydro- 
phobia department  of  the  Berlin  Institute  for  Infectious  Diseases,  the 
authorities  have  adopted  the  following  scheme,  which  stands  midway 
between  Pasteur's  classical  method  and  the  extreme  procedure  of  Ferran. 

Scheme  for  treatment  of  mild  infections: 


Day  of  injection  .  . 

i 

2 

3 

4 

S 

6 

7 

8 

9 

10 

II 

12 

** 

14 

15 

16 

17 

18 

19 

20 

21 

Number  of  days 

8-7-6 

5-4 

4-3 

5 

4 

3 

3 

a 

2 

5 

5 

4 

4 

3 

3 

2 

2 

4 

3 

2 

2 

cord  was  dried. 

Amount  injected 

0.5  of 

i.  5  of 

2.O 

3-0 

3- 

i.S 

2 

i 

I 

2 

2 

2 

2 

2 

2 

I-S 

i-5 

2 

2 

i-5 

2 

in    cubic    centi- 

each 

each 

1.0 

meters     of     an 

emulsion  i  c.cm. 

of     cord    in     5 

c.cm.  of   sterile 

bouillon. 

Scheme  for  treatment  of  severe  infections: 


i 

2 

3 

4 

5 

6 

;7 

8 

9 

10 

II 

12 

X3 

14 

IS 

16 

17 

18 

19 

20 

21 

Age  of  cord  

8-7-6 

4-3 

5-4 

3 

3 

-3. 

2 

'*•' 

5 

4 

'•4; 

3 

3' 

2 

2 

4 

3- 

2 

2 

3 

2 

Amount                               .  . 

i-5 

i-5 

i-5 

2 

2 

i 

I 

i 

2 

2 

2 

2 

2 

2 

2 

2 

> 

;2 

2 

2 

2 

ACTIVE  IMMUNIZATION 


In  severe  injuries  the  entire  treatment  is  repeated  after  one  month's  interval. 

There  is  at  present  no  doubt  whatsoever  as  to  the  value  of  these  antirabic  vaccina- 
tions. Compiled  from  a  great  number  of  statistics,  the  mortality  of  those  infected  or 
exposed  to  infection  but  untreated  is  15  to  16  per  cent.,  while  the  death  rate  of  those 
treated  at  the  Berlin  Institute  during  1898  to  1901  was  0.55  per  cent.  Similar  figures 
are  given  by  the  other  institutions. 

The  serum  of  individuals  who  have  taken  the  Pasteur  treatment  con- 
tains antibodies  that  can  neutralize  the  toxic  effects  of  the  rabies  virus. 
If  a  virulent  strain  of  the  latter  is  mixed  with  the  serum  and  injected  into 
an  animal,  no  symptoms  will  develop.  A  similar  serum  is  manufactured 
at  the  Pasteur  Institute  by  the  intravenous  injection  of  sheep  with  emul- 
sions of  the  street  virus  or  virus  fixe.  It  is  used  mainly  for  immunization 
of  animals  by  the  so-called  "  simultaneous  method/'  whereby  mixtures  of  a 
virulent  virus  and  the  serum  are  employed.  In  this  way  an  immunity  is 
attained  much  more  rapidly  than  by  the  classical  method.  A.  Marie  and 
Remlinger  have  made  use  of  this  simultaneous  method  also  in  man,  with 
good  results.  It  is  of  value  especially  when  a  rapid  immunity  is  essential 
as  in  severe  infections  (bite  of  wolf,  injuries  of  the  face)  or  neglected  cases. 
The  time  elapsed  between  the  injury  and  the  onset  of  treatment  is  an  im- 
portant deciding  factor  as  to  the  final  result.  The  following  table  of 
Diatroptoff  demonstrates  this: 


The  bitten  individual  started 
treatment  during  the 

Number  of 
patients  treated 

Of  these  died 

Percentage 

First  week 

4602 

26 

o  ^6 

Second  week  

QQI 

16 

1.66 

Third  week  . 

•217 

10 

7      IQ 

The  immunity  attained  by  vaccination  is  of  comparatively  short  duration; 

probably  only  several  years.     It  is  advisable  therefore  in  case  of  reinfection 

not   to   depend  upon  the  previous  immunity  but  go  through  another 

treatment. 

Attempts  have  been  made  to  employ  this  principle  of  virus  attenuation 
for  other  infections.  Behring  and  Koch  tried  immunization 
aSamst  bovine  tuberculosis  by  inoculation  with  living  human 

Tuberculosis  tubercle  bacilli.     These  can  be  bought  under  the  name  of 
Bcvovaccine  (v.  Behring)  and  Tauruman  (Koch). 


L 


Tauruman  is  prepared  by  the  Hochst  Farbwerke  and  is  put  up  in  sealed  glass  tubes 
which  contain  0.02  to  0.04  gm.  of  living  tubercle  bacilli  suspended  in  10  c.cm.  of 
normal  saline  solution.  This  Tauruman  is  previously  examined  in  Ehrlich's  Institute 
and  note  is  taken  of  its  purity,  quantity  of  bacteria,  virulence  against  guinea-pigs  and 
avirulence  against  rabbits  (characteristics  of  the  human  type  of  tubercle  bacilli). 


SENSITIZED   BACTERIA  3! 

To  this  class  of  experimental  work  belong  also  the  attempts  of  Fried- 
mann  to  immunize  against  human  tuberculosis  by  the  use  of  the  tubercle 
bacilli  of  cold-blooded  animals,  and  those  of  Wassermann,  Ostertag  and 
the  author,  to  inoculate  against  hog  cholera  with  living  cultures  of  mouse 
typhoid. 

Besides  the  preceding  mode  of  virus  attenuation  by  passage  through 

animals,  there  are  other  methods  employed  for  the  diminu- 

Other      tion  of  the  toxicity  of  the  virus.     Growing  the  bacteria  at 

Methods  of  too  high  a  temperature,  or  exposing  bacterial  emulsions  to 

Vaccine     light,    disinfectants   or   moderate   heating,    accomplishes  the 

Preparation.  same  purpose. 

The  mixture  of  bacteria  with  their  specific  serum  (i.e.,  serum 
obtained  from  animals  that  have  been  inoculated  with  these  bacteria), 
also  diminishes  the  virulence  of  the  bacteria.  Such  bacteria  are  des- 
ignated by  Bordet  as  " sensitized."  In  this  mixture,  the  bacteria 
attach  their  specific  antibodies;  after  'centrifugalization,  the  added 
specific  serum  now  devoid  of  its  specific  antibodies  is  removed,  and 
the  sensitized  bacteria  can  be  used  as  vaccines.  Inoculations  of  the  latter 
rarely  produce  any  infiltration.  The  same  object  can  be  accomplished 
by  injecting  bacteria  and  at  the  same  time  also  their  specific  serum.  This 
is  technically  simpler  and  is  known  as  the  "Simultaneous  Method."  It 
has  shown  itself  of  great  value  in  Lorenze's  prophylactic  inoculations 
against  swine  erysipelas. 

2.  Immunization  with  Dead  Bacteria. — Immunization  with  dead 
bacteria  was  first  undertaken  by  Toussaint,  Salmon  and  Smith,  and 
Chamberland  and  Roux. 

This  method  is  to  be  distinctly  separated  from  those  already  discussed.  Bail 
claims  that  the  immunization  with  living  bacteria  as  well  as  by  aggressins  (to  be 
mentioned  later)  is  an  immunization  against  the  infectious  disease;  while  the  immuniza- 
tion with  dead  bacteria  is  an  immunization  against  the  bacterial  bodies.  While  this 
holds  true  for  some  bacteria,  it  is,  to  say  the  least,  questionable  whether  it  can  be 
considered  a  general  rule. 

Whenever  a  real  immunity  is  desired — that  is,  protection  against 
disease,  a  vaccine  either  in  the  form  of  living  or  attenuated  bacteria  should 
be  given  the  preference.  Up  to  a  certain  degree,  the  extracts  of  living  bac- 
teria, and  the  natural  and  artificial  aggressins  can  be  similarly  employed. 
If,  however,  no  real  immunity,  but  just  a  serum  containing  a  great  number 
of  antibodies  is  wanted,  as  in  serum  diagnosis,  for  agglutination,  bacteri- 
olysis, complement  fixation,  etc.,  then  immunization  by  dead  bacteria 
is  just  as,  if  not  more  so,  efficient. 

Recently,  the  question  has  been  raised  whether  the  antibodies  pro- 
duced by  immunization  with  heated  antigens  are  identical  with  those  ob- 
tained with  unheated  antigens.  The  experiments  of  Obermeyer  and  Pick, 


32  ACTIVE  IMMUNIZATION 

which  will  be  referred  to  under  proteid  immunization,  seem  to  prove  that 
they  are  not  alike.  For  laboratory  work  it  is  advisable  to  use  living 
cultures  only  in  cases  of  absolute  necessity. 

In  heating  bacteria  to  destroy  their  virulence  and  thus  be  suitable  for 
inoculation,  we  must  be  very  careful  not  to  raise  the  tempera- 
Death  of  ture  to  such  a  degree  that  not  only  the  toxicity  but  also  the 
Bacteria  immunization  power  is  destroyed.  It  is  best  to  employ  the 
by  Heat,  minimum  amount  of  heat  which  will  kill  the  respective  bacteria. 
For  most  of  these  as  Typhoid,  Paratyphoid,  Colon,  and  Dysen- 
tery bacilli,  Cholera  Vibrios,  Meningo-,  Staphylo-,  Strepto-  and  Pneumo- 
cocci,  one  hour  at  60°  C.  is  sufficient. 

The  bacteria  are  grown  upon  agar  cultures  and  the  required  amount 
is  removed  and  suspended  in  sterile  physiological  salt  solution  or  bouillon. 
This  suspension  is  then  placed  into  a  hot  water  bath  or  thermostat  regu- 
lated at  60°  C.,  for  one  hour.' 

If  the  bacteria  employed  are  highly  infectious,  one  must  be  sure  that 
all  bacteria  have  been  killed.  This  must  especially  be  noted  when  giving 
prophylactic  inoculations  in  man.  Several  drops  of  the  emulsion  are 
therefore  transferred  to  agar  tubes  and  incubated  for  a  day  or  two.  If 
a  growth  appears,  the  emulsion  is  to  be  reheated;  if  not  it  can  be  considered 
sterile. 

The  mode  of  immunization  with  dead  bacteria  is  the  same  as  has 
been  described  for  the  living  ones.  In  general  the  dosage  to  be  used  may 
be  larger. 

Small  doses  are  injected  at  first,  followed  later  on  by  increasing  quanti- 
ties at  intervals  of  five  to  eight  days,  e.g. 

Intravenous  inoculation  of  a  rabbit  with  dead  typhoid  bacilli. 

Result. — Protection  against  living  virulent  bacteria,  appearance  of 
agglutinins,  bacteriolysins,  bacteriotropins  and  complement  binding 
substances  in  the  serum. 

i ./I.  1909.     Rabbit  No.  i.     i  loopful  of  a  typhoid  agar  slant  culture  killed  at  60°  C. 

and  injected  intravenously. 
6./I.  4  loopfuls  of  typhoid  culture  killed  at  60°  C.  and  injected 

intravenously. 

I2./I.  i  culture  of  typhoid  killed  at  60°  C.  and  injected  intra- 

venously. 
20./I.  Infection  with  i  culture  of  the  living  typhoid  bacilli 

injected  intravenously.     Animal  remains  alive. 
Rabbit  No.  2.     Control. 

20./I.  Infection:  1/4  loopful  of  living  typhoid  bacteria  intra- 

venously. 
22./I.  f(death). 

The  use  of  killed  typhoid  bacteria  for  prophylactic  immunization  has 
recently  been  widely  adopted.  This  has  been  stimulated  to  a  great  degree 
by  the  successful  experiments  of  Wright,  and  Pfeiffer  and  Kolle. 


PROPHYLACTIC    TYPHOID    INOCULATION  33 

Wright's  Method  of  Prophylactic  Typhoid  Inoculation. 

The  vaccine  originally  employed  by  Wright  for  these  inoculations  con- 
sisted of  highly  virulent  cultures  of  Bacillus  Typhosus  grown  in  broth  for 
twenty-four  to  forty-eight  hours  (sometimes  even  for  four  weeks),  and 
sterilized  by  heating  at  60°  C.  The  vaccine  was  then  standardized,  i.e., 
the  strength  of  the  vaccine  was  fixed  in  accordance  with  another  of  known 
strength,  the  dosage  of  which  had  been  gauged  by  inoculations  in  man. 
The  early  form  of  standardization  consisted  in  determining  the  toxicity 
of  the  virus.  Guinea-pigs  weighing  250  to  300  gms.  were  inoculated 
subcutaneously  with  0.5,  0.75,  i.o  and  1.5  c.cm.  of  the  vaccine  respec- 
tively. Death  to  some  of  the  animals  would  come  in  twelve  hours  to 
three  days.  The  amount  required  to  kill  a  guinea-pig  weighing  100  gms. 
or  rather  the  proportional  fraction  of  the  dose  which  proved  fatal  to  the 
one  of  250  to  300  gms.  was  taken  as  the  standard  dose  for  injection  in 
man.  Wright  subsequently  found  that  better  results  were  obtained,  if  the 
vaccine  was  prepared  from  twenty-four  hour  cultures  grown  upon  the  sur- 
face of  agar.  The  growth  is  then  washed  off  in  physiological 
Preparation,  saline  solution.  This  emulsion  is  sterilized  by  subjecting 
it  to  a  temperature  of  55°  to  60°  C.  for  one  hour,  after  which 
the  number  of  bacteria  is  computed  by  Wright's  method  (see  Standard- 
ization of  Vaccines  in  chapter  on  Opsonins).  After  this  the  emulsion  is 
further  diluted  with  physiological  salt  solution  containing  0.5  per  cent, 
carbolic  or  0.25  per  cent,  trikresol  in  such  a  manner  that  two  or  three 
different  concentrations  are  secured,  one  containing  500  million  killed 
typhoid  bacilli  per  c.cm.,  one  containing  1000  million  per  c.cm.,  and  one 
containing  2000  million  per  c.cm. 

The  particular  strain  of  typhoid  bacillus  employed  for  the  vaccine 
varies.  Some  use  a  strain  isolated  by  Leishman  in  1900.  This  is  selected 
not  on  account  of  its  degree  of  virulence  but  on  account  of  its  property 
of  being  able  to  stimulate  the  formation  of  a  great  amount  of  antibodies. 
The  editor  and  others  employ  a  mixture  of  eight  or  ten  different  strains 
with  perhaps  one  or  two  paratyphoid  strains. 

The  typhoid  vaccine  or  typho-bacterin  as  it  is  frequently  called  is 
administered  subcutaneously,  usually  in  the  arm  at  the  insertion  of  the 
deltoid  muscle.  The  needk  should  not  enter  the  muscle  or  find  its  way 
between  the  layers  of  the  skin.  The  arm  should  be  cleansed  as  for  any 
other  injection  with  alcohol  and  iodine. 

The  dosage  almost  uniformly  employed  consists  of  500  million 
Dosage,      bacteria  for  the  first  injection  and  1000  million  for  each  of  the 
two  subsequent  injections  at  intervals  of  eight  to  ten  days. 
If,  however,  such  an  extended  period  of  time  is  not  available,  then  two  in- 
oculations will  suffice,  the  first  dose  1000  million  and  the  second  dose 


34  ACTIVE  IMMUNIZATION 

2000  million.  Wright  used  the  two  larger  doses.  An  objection 
raised  to  this  method  is  that  the  general  reaction  obtained  is  more  severe. 
The  editor,  however,  has  employed  these  larger  doses  for  inoculating  the 
nursing  and  medical  staff  of  the  hospital  and  a  great  number  of  laymen, 
without  any  ill  effect. 

Women  and  children  should  receive  a  dose  in  proportion  to  their 
weight;  a  healthy  man  weighing  150  pounds  being  designated  as  the  stand- 
ard of  comparison. 

Local  and  general  reactions  follow  the  inoculations.  Thus  local  redness 
and  swelling  of  the  skin,  lymphangitis  and  enlargement  of  the  neighbor- 
ing glands  are  the  usual  consequences.  The  inflammation  can  at  times 
be  severe  enough  to  simulate  erysipelas.  The  general  symptoms,  on 
the  other  hand,  may  consist  of  a  general  feeling  of  illness,  headache, 
little  fever,  and  occasionally  nausea,  not  infrequently  accompanied  by 
vomiting.  These  signs  of  indisposition,  however,  pass  off  rapidly  without 
leaving  any  permanent  ill  effects.  Debilitated  persons  frequently  present 
the  most  profound  reactions.  Occasionally  latent  and  chronic  diseases 
of  a  non-typhoidal  character  may  be  made  active  by  inoculation.  These 
exacerbations  are  not  serious  and  usually  by  diminishing  the  quantity 
and  increasing  the  number  of  doses,  these  effects  can  be  avoided.  Six 
to  eleven  days  after  the  injection,  an  increase  in  the  number  of  agglutinat- 
ing, bacteriolytic  and  bacteriotropic  bodies  can  be  demonstrated  in  the 
blood  of  the  inoculated  individual.  The  immune  bodies  reach  their 
height  in  two  to  three  months  after  incculation,  and  then  fall 
rapidly.  In  a  series  of  forty  cases  Garbat  was  unable  to  demonstrate 
complement  fixation  bodies  with  any  regularity  in  the  blood  after  pro- 
phylactic injection. 

As  to  the  results  of  antityphoid  vaccination,  opinion  is  somewhat 
divided.  According  to  Wright's  statistics  infections  have  been  diminished 
by  about  one-half,  and  in  single  series  to  one-sixth  or  even  one-twenty- 
eighth  of  the  former,  or  control  number.  The  mortality  too  is  much  lower. 
Out  of  1758  individuals  who  had  been  vaccinated,  only  142  or  8  per 
cent,  died;  out  of  10,980  who  had  not  been,  1800  or  16.6  per  cent,  met 
death. 

The  immunity  attained  is  not  absolute,  for  according  to  Russel,  in 
1911,  among  80,000  p.ersons  vaccinated  in  the  United  States  Army  there 
were  twelve  cases  of  typhoid  with  one  death  (due  to  intestinal  hemor- 
rhage), and  in  1910  six  cases  occurred  with  no  fatalities.  Had  it  not  been 
for  the  prophylactic  immunization,  there  would  have  occurred  at  the  pre- 
vailing rates  of  incidence  about  250  cases.  Similar  favorable  statistics 
have  been  collected  in  England,  Germany  and  France.  The  period  of 
immunity  lasts  from  two  to  three  years. 


PROPHYLACTIC    TYPHOID   INOCULATION  35 

Pfeiffer-Kolle's  Experiments. 

Pfeiffer  and  Kolle  prepare  their  vaccine  by  growing  typhoid  bacilli 
on  agar  cultures  and  suspending  a  twenty-four  hours'  growth  in  physio- 
logical NaCl  solution.  The  normal  platinum  loop  is  the  unit  of  standard- 
ization. A  full  grown  agar  culture  is  considered  as  10  normal  loops  and 
as  such  it  is  diluted  in  4.5  c.cm.  of  saline.  This  emulsion  is  placed  in 
a  thermostat  at  60°  C .  for  two  hours  and  then  tested  for  its  sterility.  Suffi- 
cient 5  per  cent,  phenol  solution  is  next  added  to  the  suspension  to  bring 
the  contents  up  to  a  0.5  per  cent,  carbolic  solution,  and  the  final  emulsion 
is  again  heated  at  60°  C.  for  thirty  minutes.  One  c.cm.  of  the  vaccine 
is  thus  equivalent  to  two  normal  loops  of  culture.  The  amounts  of 
vaccine  to  be  injected  have  not  yet  been  definitely  decided  upon.  The 
best  dosage  so  far  is  the  following: 

For  the  first  injection:  0.3  c.cm.  of  the  vaccine. 
For  the  second  injection:  0.8  c.cm.  of  the  vaccine. 
For  the  third  injection:  i.o  c.cm.  of  the  vaccine. 

The  injection  is  made  subcutaneously  between  the  breast  and  clavicle. 

The  local  and  general  reactions  are  the  same  as  those  observed  with 
Wright's  method.  As  a  result  of  the  injection  only  increased  agglutinins 
and  bacteriolysins  have  been  found  in  the  blood  serum.  Bacteriotropins 
have  not  as  yet  been  examined  for. 

The  effects  of  these  inoculations  seem  to  be  very  good.  Protection  is  prolonged 
according  to  the  increase  in  the  number  of  injections,  and  if  inoculated  individuals  do 
become  infected,  they  run  a  very  much  milder  course  of  the  disease. 

The  following  statistics  as  given  by  Kuhn  indicate  the  results: 

Inoculated.  Non-inoculated. 

Very  slightly  ill 186  (50. 13  per  cent.)  331  (36. 55  per  cent.) 

Moderately  ill 96  (25.88  per  cent.)  225  (24.85  per  cent.) 

Severely  ill 65  (17.52  per  cent.)  234  (25,80  per  cent.) 

Deaths 24  (  6.47  per  cent.)  116  (12.80  per  cent.) 


371  (100      per  cent.)  906  (100      per  cent.) 

The' prophylactic  immunity  according  to  Kuhn  lasts  one  year.     Kolle  has  under- 
taken similar  experiments  against  cholera. 


CHAPTER  IV. 
ACTIVE  IMMUNIZATION. 

Immunization  with  Bacterial  Extracts.— Aggressin  Experiments. 

The  marked  infectious  nature  of  the  organisms  belonging  to  the  class 
of  "pure  parasites"  makes  it  very  difficult  to  produce  an  immunity  against 
them.  They  possess  no  sublethal  dose  in  their  living  state,  and  if  used 
when  dead,  will  produce  no  prophylactic  immunity.  By  artificial  atten- 
uation of  these  living  virulent  bacteria  Pasteur  succeeded  in  obtaining 
vaccines  of  several  of  them.  The  methods  that  he  employed  were,  however, 
totally  impracticable,  for  not  infrequently,  by  the  use  of  the  vaccine,  the 
disease  which  it  was  the  object  to  prevent  was  instigated.  It  was  there- 
fore a  distinct  and  important  triumph  when  Bail  and  Weil  showed  that 
immunity  against  these  parasites  could  be  attained  by  using  as  vaccine 
antigen,  the  so-called  "aggressins";  i.e.,  exudates  from  animals  that  had 
been  infected  with  the  respective  bacteria. 

Bail's  explanation  of  the  aggressin-immunization  method  is  entirely  theoretical. 
He  believes  that  during  an  infection,  the  bacteria  secrete  certain  agents  which  counter- 
act or  entirely  destroy  the  infected  organism's  protective  powers,  especially  phago- 
cytosis. These  bodies  he  called  aggressins  and  they  were  distinguished  by  the  fact 
that  they  were  formed  by  living  bacteria,  and  only  in  the  living  body.  According  to 
Bail,  the  pathogenicity  of  bacteria  depends  upon  their  power  to  produce  these  aggres- 
sins. If  this  theory  be  correct,  it  should  be  possible  to  demonstrate  aggressins,  espe- 
cially in  infections  where  the  protective  power  of  the  organism  is  almost  nil,  as  for 
example  an  infection  produced  by  the  bacteria  belonging  to  the  group  of  hemorrhagic 
septicemia.  Unfortunately,  in  actual  practice  this  is  not  so. 

The  following  experiment  gives  an  idea  of  the  true  nature  of  these 
aggressins  and  how  they  are  obtained. 

At  first,  an  infecting  agent — the  bacillus  of  swine  pest,  may  be  chosen. 
This  micro-organism  belongs  to  the  same  class  as  chicken  cholera  and  fowl 
plague,  and  is  distantly  related  to  the  human  pest.  For  rabbits,  this 
bacillus  is  a  pure  parasite,  for  giunea-pigs,  by  subcutaneous  inoculation, 
a  half  parasite. 

The  Obtaining  of  Aggressins. 

One  drop  of  a  twenty-four-hour  brcth  culture  of  this  swine  pest  bacillus, 
in  5  c.cm.  bouillon,  is  injected  intrapleurally  in  a  rabbit  in  the  following 
manner. 

36 


OBTAINING   NATURAL   AGGRESSINS 


37 


A  small  incision  is  made  in  one  of  the  intercostal  spaces  on  the  side 
of  the  chest,  and  through  this  wound  a  long  canula  is  introduced  into  the 
pleural  cavity.  The  animal  as  a  rule  rapidly  succumbs  to  the  infec- 
tion. On  autopsy,  the  pleural  cavity  is  found  to  contain  an  exudate  of 
a  reddish-brown  color  (hemorrhagic)  on  the  side  where  the  inoculation 
was  given,  and  of  yellow  serous  fluid  on  the  other  side.  The  bloody  exu- 
date, about  15  c.cm.,  is  removed  with  a  sterile  pipette,  placed  in  a  sterile 
centrifuge  tube  to  which  is  added  1.5  c.cm.  of  5  per  cent,  carbolic  acid 
drop  by  drop  (making  the  entire  solution  a  1-2  per  cent,  carbolic  acid 
dilution),  agitated  continually  in-  order  to  prevent  precipitation,  and 
followed  by  centrifugalization  at  a  high  speed  for  many  hours  until  it 
becomes  very  clear.  The  upper  clear  part  which  is  now  free  of  bacteria, 
or  very  nearly  so,  is  pipetted  off  and  heated  for  three  hours  at  44°  C.  Its 
sterility  is  then  tested  and  if  no  growth  appears  after  forty-eight  hours,  it 
is  considered  sterile. 


First  Fundamental  Aggressin  Test. 

(Its  Power  of  Increasing  Severity  of  Infections.) 


No. 

Animal 

Date 

Amount  cf  infective 
material 

Aggressins 

Result 

i 

Guinea-pig. 

6/IV'os. 

i/ioo  loopful  of  swine 

Remains  alive. 

pest  subcutaneously. 

2 

Guinea-pig. 

6/IV'os. 

i/  100  loopful  of  swine 

+  1.5  c.cm. 

t  on  third  day. 

pest  subcutaneously. 

of  aggressins 

subcutaneously. 

3 

Guinea-pig. 

6/IV'o5. 

-j-  1  .  5  c.cm. 

Remains  alive. 

of  aggressins 

subcutane- 

ously. 

4 

Guinea-pig. 

6/IV'o5. 

i/ioo  loopful  of  swine 

+3  c.cm.  sub- 

f on  second  day. 

pest  subcutaneously. 

cutaneously. 

5 

Guinea-pig. 

6/IV'o5. 

'.  

3  c.cm.  subcu- 

Remains alive. 

taneously. 

6 

Guinea-pig. 

6/IV'os. 

i/  1000    loopful    subcu- 

-f-2 c.cm.  sub- 

On 7/IV  very  ill; 

taneously. 

cutaneously. 

on  8/1  V  very  ill; 

9/IV    very    ill; 

zo/IV  very  thin; 

i  |  V  m~  : 

•  ;  ' 

n/IV  begins  to 

pick    up    slowly 

and    remains 

alive.      Marked 

infiltration 

around  point  of 

injection. 

38  ACTIVE  IMMUNIZATION 

It  can  be  deduced  from  this  experiment  that  i/ioo  of  a  loopful  of  swine 
pest  culture,  which  represents  i/io  of  a  fatal  dose  for  a  guinea-pig  by  sub- 
cutaneous injection,  can  be  converted  into  an  acutely  fatal  dose  by  injecting 
the  aggressin  simultaneously  or  a  half  hour  before  the  swine  pest  culture. 

The  aggressin  itself  is  only  slightly  toxic,  and  the  quantity  injected  is 
well  borne  by  the  guinea-pig.  Its  power  of  increasing  the  virulence  of  the 
infective  material  varies  directly  with  its  quantity,  i.e.,  the  greater  the  dose 
of  aggressin,  the  more  rapidly  is  death  occasioned.  If,  however,  only  small 
doses  of  the  culture  are  given,  and  in  addition  to  this,  the  aggressin  is  in- 
jected, the  animal  does  not  die,  but  becomes  exceedingly  ill,  thus  indicating 
the  effect  of  aggressins.  In  this  connection  it  might  be  well  to  add  that 
the  aggressin  may  be  given  twenty-four  hours  previous  to  the  time  of 
infection. 

On  microscopical  examination  of  the  aggressin  exudate,  only  very  few 
cells,  but  a  great  number  of  bacteria  are  present.  The  bacteria  here  have 
increased  during  the  short  time  after  the  infection  to  a  far  greater  extent 
than  they  would  have  done  in  an  artificial  medium.  The  body,  continu- 
ally in  combat  against  their  increasing  toxicity,  finds  itself  powerless  when 
its  limited  fighting  capacity,  decreasing  in  proportion  to  the  rise  in  strength 
of  the  hostile  micro-organisms,  is  expended;  and  ultimately  succumbs  to 
the  infection.  During  the  struggle  between  the  protective  forces  of  the 
organism  and  the  invading  bacteria,  many  of  the  latter  are  destroyed  and 
these  disintegrated  bacteria  are  found  within  the  exudate.  From  this  fact 
Wassermann  and  Citron  formed  the  conclusion  that  the  aggressins  are  not 
as  Bail  claimed,  secretory  products  of  live  bacteria  produced  during  the 
conflict  between  the  bacteria  and  the  body  organism,  but  rather  the  prod- 
ucts of  broken  down  bacteria.  Therefore,  Bail's  supposition  that  aggres- 
sins are  only  obtained  in  the  living  body  is  erroneous  and  can  be  shown  to  be 
so  by  the  fact  that  aggressins  may  be  reproduced  whenever  the  essential 
requirements  can  be  had,  and  these  are: 

1.  Large  numbers  of  bacteria. 

2.  Non-poisonous  agents  which  can  disintegrate  these  bacteria. 
Aggressins  thus  obtained  are  known  according  to  Wassermann  and 

Citron,  as  "artificial"  in  contrast  to  Bail's  " natural"  ones. 

Wassermann  and  Citron  Method  of  Obtaining  Artificial  Aggressins. 

Cultures  are  grown  in  mass  on  Kolle's  flask'plates.  A  Kolle's  agar 
plate  is  equivalent  to  twelve  agar  slants.  For  the  inoculation  of  these 
flasks  a  long  platinum  loop  is  needed  which  transfers  some  of  the  culture  to 
the  plate.  The  transferred  material  is  then  spread  over  the  entire  surface 
of  the  flask  by  a  large  triangular  platinum  loop.  The  latter  is  made  by  in- 
serting into  a  holder  both  ends  of  a  not  too  thin  platinum  wire,  about  20 


OBTAINING   ARTIFICIAL   AGGRESSINS  39 

cm.  in  length  which  is  then  shaped  into  a  triangular  form.  While  still 
red  hot,  this  triangular  loop  should  be  introduced  in  the  flask  and  allowed 
to  cool  there.  Before  the  culture  is  spread,  it  is  advisable  to  bend  the 
entire  loop  to  a  slight  angle  by  pressing  it  against  the  upper  wall  of  the 
flask,  thereby  preventing  the  hot  end  of  the  loop  holder  from  coming  in 
contact  with  the  agar  surface.  It  is  best  also  to  test  the  platinum  loop 
upon  the  surface  of  the  agar  in  order  to  ascertain  whether  it  is  still  too  hot. 

After  twenty-four  hours  of  incubation  there  is  usually  a  pronounced 
growth  upon  the  plates.  This  culture  is  then  washed  off  either  by  serum 
or  distilled  water  (" serous"  or  " aqueous  aggressin").  The  former  may 
be  obtained  fresh  from  a  rabbit.  Usually  10  to  12  c.cm.  of  fluid  per  flask 
is  required;  3  or  4  c.cm.  are  first  poured  upon  the  culture  growth  and  the 
mass  scraped  gently  but  quickly  with  the  triangular  loop.  Then  the 
remainder  of  the  fluid  7  to  8  c.cm.  is  poured  in  to  release  the  still  adherent 
bacteria.  The  turbid  milky  emulsion  is  collected  either  in  a  small  dark 
glass  Erlenmeyer  flask  or  a  brown  bottle.  This  is  then  placed  into  a  proper 
apparatus  and  shaken  for  one  to  two  days  at  room  temperature.  Enough 
5  per  cent,  carbolic  acid  is  added  to  make  a  1/2  per  cent,  phenol  solution, 
and  the  emulsion  is  centrifugalized  and  sterilized  in  the  same  manner  as 
has  been  described  for  the  natural  aggressins. 

The  tendency  of  aggressins  toward  increasing  virulence  ("infektions 
bef orderung  ")  is  the  same  whether  these  aggressins  are  artificial  or  natural. 

From  the  following  experiment  it  can  be  seen  that  the  bacteria  contain 
some  substance  which  is  easily  soluble  in  the  body  fluids  and  in  distilled 
water,  and  which  increases  the  infectious  nature  of  the  respective  bacteria 
when  injected  simultaneously  with  them.  In  small  doses,  this  substance 
is  not  poisonous,  in  large  doses  it  may  be,  but  is  not  necessarily  so.  There 
is  no  definite  relation  between  the  poisonous  qualities  of  the  aggressin  and 
its  power  to  increase  the  virulence  of  an  infection.  This  disproves  the  as- 
sumption of  some  authors  that  the  action  of  the  aggressins  is  dependent 
upon  the  toxicity  of  the  endotoxins. 


ACTIVE   IMMUNIZATION 


Experiment. 


No.         Animal 

Date 

Amount  of  in- 
fective material 
from  agar  cul- 
ture 

Serous 
aggressin 

Watery 
K.( 

aggressin 

j 
i        Guinea-pig.     2Q/V  '05. 

i/  200   loop   of 

i 
Re 

Results 


I 

2 

3 
4 

5 

6 

7 
8 

Guinea-pig. 
Guinea-pig. 
Guinea-pig. 
Guinea-pig. 
Guinea-pig. 

Guinea-pig. 
Guinea-pig. 
Guinea-pig. 

2Q/V  '05. 
29/V  '05. 

2Q/V    '05. 
2/VI  '05. 
2/VI   '0S. 

2/VI   '05. 
2/VI  '05. 
2/VI  '05. 

I/2OO     loop     Of 

swine  pest  sub- 
cutaneously. 
i/  200    loop  ot 
swine  pest  sub- 
cutaneously. 

i/  200  loop  of 
swine  pest  sub- 
cutaneously. 
1/200  loop  of 
swine  pest  sub- 
cutaneously. 

-j-2  5  c  cm 

Remains 
alive. 

f  after 
twenty-four 
hours 
Remains 
alive. 

f  after  three 
days. 

1  f  after  three 
days. 

Remains 
alive, 
t  in  twenty- 
;    four  hours. 
Remains 
alive. 
1          .      ; 

subcutane- 
ously. 
-{-2  5  c  cm. 

subcutane- 
ously. 
2  c.cm.  subcu- 
taneously. 

3  c.cm.  subcu- 

taneously. 
.    .    .  .    3  c.cm.  subcu- 

1/200  loop  of 
swine  pest. 

taneously. 
4.5  c.cm.  sub- 
cutaneously. 

Second  Fundamental  Aggressin  Test. 

(Its  Property  of  Active  Immunization.) 

Bail  and  his  pupils  believe  that  when  bacteria  invade  a  normal  organ- 
ism, it  is  the  aggressin  power  of  these  bacteria  which  determines  whether  or 
not,  by  their  multiplication,  disease  will  set  in.  If  infection  does  take 
place,  it  continues  until  the  "aggressive"  nature  of  the  bacteria  is  curbed. 
As  there  are  some  bacteria  which  on  injection  do  not  produce  any  disease, 
Bail  attributes  this  phenomenon  of  immunity  to  the  missing  " aggressive" 
action  of  the  respective  bacteria.  It  is  not  merely  the  presence  of  bacteria 
which  is  the  criterion  for  the  existence  of  disease;  as  long  as  they  are  void 
of  their  " aggressive"  property,  they  have  actually  become  saprophytes. 

Accordingly,  Bail  believes  that  the  bactericidal  immunity  is  no  true 
immunity  because  it  can  be  obtained  by  injection  of  dead  micro-organisms 
or  by  live  bacteria  in  such  minute  doses  that  no  specific  symptoms  are  pro- 
duced, i.e.,  no  aggressins  are  produced  within  the  body.  "If  the  im- 
munity lacks  the  "anti-aggressive"  component,  which  alone  governs  the 
existence  of  disease,  one  gains  only  an  apparent  immunity  against  the  ex- 
citing factor  of  the  disease,  but  not  against  the  disease  itself" 


IMMUNIZATION   WITH   NATURAL   AGGRESSINS  41 

Bail  places  the  utmost  stress  upon  the  difference  between  an  immunity 
directed  against  the  exciting  agent  of  the  disease  (bactericidal  immunity)  and 
that  against  the  disease  itself  (anti-aggressive  immunity). 

Immunization  against  the  disease  is  only  possible  if  the  aggressin  reaches  the  body 
of  the  animal  to  be  immunized.  This  is  possible  either  by  employing  Pasteur's  method 
of  vaccine  inoculation,  i.e.,  the  injection  of  bacteria,  the  "aggressive"  nature  of  which 
has  been  weakened  but  not  destroyed,  or  by  direct  inoculations  of  aggressins.  The 
latter  is  by  far  the  simpler  and  more  reliable  mode  of  procedure,  being  productive  of 
a  true  immunity. 

s 
Nowhere  does  this  problem  appear  of  such  extreme  importance  as 

where  immunity  against  a  pure  parasite  is  contemplated,  as  in 

Immunity    the  case  of  swine  pest  and  chicken  cholera.     While  it  is  ex- 

^air          ceedingly  difficult,  in  fact  almost  impossible  to  immunize 

Parasite       against  these  bacteria  either  with  dead  or  living  germs  or 

vaccines,  this  task  is  readily  accomplished  by  the  injection  of 

non-poisonous  aggressins,  ina  smuch  as  they  are  well  tolerated.     In  addition, 

these  bacteria  are  of  help  in  definitely  deciding  whether  or  not  an  aggressin 

immunity  is  at  all  possible. 

Weil,  a  co-worker  of  Bail's,  has  carried  out  these  experiments  for  chicken 
cholera,  while  the  author  has  done  the  same  for  swine  pest. 


Example  of  Active  Immunization  with  Natural  Aggressins. 

a.  Slow  Immunization. 

Rabbit  I. 

6./IV.  1905  ist  injection:  i.o  c.cm.  natural  swine  pest  aggressin  intraperitoneally. 

ly./IV.  2d  injection:  i.o  c.cm.  natural  swine  pest  aggressin  intraperitoneally. 

25./IV.  3d  injection:  i.o  c.cm.  natural  swine  pest  aggressin  intraperitoneally. 

i./V.  4th  injection:  2.0  c.cm.  natural  swine  pest  aggressin  subcutaneously. 

I2./V.  5th  injection:  2.0  c.cm.  natural  swine  pest  aggressin  subcutaneously. 

i6./VI.  ist  infection;  with  i/ioo  loopful  of  swine  pest  culture  intravenously. 

i y./ VI.  Perfectly  weU. 

8./VII.  2d  infection;  with  i  loopful  of  swine  pest  culture  intravenously. 

I5./VII.  Perfectly  well. 

22./IX.  3d  infection;  with  i  loopful  of  swine  pest  culture  intravenously. 

3./X.  Perfectly  well. 

Rabbit  I.  Controls.  Rabbit  II. 


i6./VI.  1905  1/100,000  loopful  of  swine 
pest  culture  intra- 
venously. 

i  y./ VI.  f  found  dead. 


8./VII.  1905  1/10,000  loopful  of  swine 
pest  culture  subcuta- 
neously. 

9./VII.  f  found  dead. 


42  ACTIVE  IMMUNIZATION 

b.  Rapid  Immunization. 

Rabbit  II. 

8./IV.     1905  Injection  of  4  c.cm.  of  natural  swine  pest  aggressin  subcutaneously. 
lo./IV.  Animal  shows  slight  ill  effects. 

I3-/IV.  Perfectly  active. 

26./IV.  ist  infection:  i/io  loopful  of  swine  pest  culture  subcutaneously. 

i6./VL  2d  infection:  i  loopful  of  swine  pest  culture  intravenously. 

Animal  remained  active. 

Controls. 

Rabbit  III. 

26./IV.     1905  1/10,000  loopful  of  swine  pest  culture  subcutaneously. 
27./IV.  f. 

These  experiments  prove  conclusively  that  by  the  method  described 
above  it  is  possible  to  attain  a  high  grade  of  immunity.  In  this  connection, 
however,  it  is  very  important  to  adhere  to  what  Bail  pointed  out,  namely, 
that  a  long  period  should  elapse  between  the  last  inoculation  with  the  aggressin 
and  the  first  injection;  the  reason  for  that  being,  that  during  the  period  of  im- 
munization, and  following  it  for  a  long  time,  there  is  a  condition  of  hyper- 
susceptibility  to  infection. 

Example  of  Active  Immunization  with  Artificial  Aggressins. 

Rabbit  i. 

3-/VI.     1905  ist  injection:  4  c.cm.  of  watery  extract  of  swine  pest  bacilli  subcutane- 
ously. 

I4./VI.  2d  injection:  2  c.cm.  of  watery  extract  of  swine  pest  bacilli  subcutane- 

ously. 

2 5.7 VI.  Removal  of  some  blood. 

4./VII.  3d  injection:  3  c.cm.  of  watery  extract  of  swine  pest  bacilli  subcutane- 

ously. 

2I./VII.  Infection:  i/io  loopful  of  swine  pest  culture  subcutaneously. 

3./X.  Animal  alive  and  healthy. 

Rabbit  2. 

ig./VI.     1905  ist  injection:  2.5  c.cm.  of  serous  extract  of  swine  pest  bacilli  subcutane- 
ously. 

9./VII.  2d  injection:  2.0  c.cm.  of  serous  extract  of  swine  pest  bacilli  subcutane- 

ously. 

I2./VII.  3d  injection:  4.0  c.cm.  of  serous  extract  of  swine  pest  bacilli  subcutane- 

ously. 

24./VII.  ist  infection:  i/io  loopful  of  swine  pest  culture  subcutaneously. 

22./IX.  2d  infection:  i  loopful  of  swine  pest  culture  intravenously. 

Animal  remains  perfectly  well. 
Rabbit  3. 

i6./VI.     1905  Injection:  2.5  c.cm.  of  watery  extract  of  swine  pest  bacilli  subcutane- 
ously. 


THE   AGGRESSIVE   IMMUNITY  43 

4./VII.  Infection:  i/ioo  loopful  of  swine  pest  culture  subcutaneously. 

I5./VIL  Small  local  infiltrate.     Very  active. 

3-/X.  Animal  alive  and  perfectly  well. 

Control  animals  inoculated  on  the  days  of  infection  died  within  twenty-four  hours 
after  inoculations  of  1/100,000  loopful  of  culture  intravenously,  1/10,000  loopful 
subcutaneously. 

It  is  evident  from  the  above,  that  an  immunity  against  pure  parasites 
can  be  obtained  just  as  well  by  one  or  several  injections  of  extracts  of  living 
bacteria,  as  by  injections  of  natural  aggressins.  Since  the  production 
of  aggressins  by  a  struggle  between  the  bacteria  and  distilled  water 
can  be  excluded,  it  can  be  taken  without  further  explanation  that  in  the 
development  of  those  substances  which  have  a  tendency  to  increase  the 
virulence  of  bacteria,  or  which  can  be  used  to  produce  an  immunity,  the 
bacteria  play  a  passive  role,  in  that  they  are  only  extracted  by  the  dis- 
solving agent.  The  difference  between  the  anti-bacterial  and  anti-aggres- 
sin  immunity  is  therefore  not  a  qualitative  one,  as  in  both  instances  they 
are  the  substances  that  are  set  free  from  the  bacteria  which  stimulate  the 
formation  of  antibodies.  When  living  virulent  bacteria  are  injected  for  the 
purposes  of  immunization,  they  increase  so  rapidly  that  a  proper  dosage  is 
impossible  and  the  animals  frequently  die  before  enough  antibodies  are 
liberated.  In  addition,  antibodies  are  also  generated  against  the  capsule 
of  the  bacteria  (bacteriolysins) . 

The  only  difference  between  immunization  with  morphologically  well 
preserved  but  dead  bacteria  and  that  with  aggressins  is  that  within  the  latter 
the  bacterial  substances  which  tend  to  bring  about  the  immunity  have  not 
been  altered  by  previous  heating,  but  exist  in  their  natural  easily  absorb- 
able  form.  Moreover,  by  using  the  extracts  one  does  away  with  certain 
toxic  substances  which  are  found  within  the  bacterial  capsules,  and  which 
are  rather  toxic  to  subcutaneous  tissue,  producing  necrosis  and  marasmus. 

The  Third  Fundamental  Aggressin  Experiment. 

Here,  it  is  demonstrated  that  the  serum  of  animals  immunized,  by  aggres- 
sins either  artificial  or  natural,  contain  antibodies  which  (i)  can  neutralize 
thai-property  of  aggressins  whereby  they  increase  the  virulence  of  bacteria; 
(2)  produce  a  passive  immunity  against  infection  with  living  bacteria. 

As  for  the  biological  structure  of  these  antibodies,  or  anti- aggressins  as 
they  may  be  called,  it  may  be  said  that  they  belong  to  the  class  cf  ambo- 
ceptors,  shown  by  the  complement  fixation  methods. 

The  practical  employment  of  aggressins  as  a  method  of  immunization 
offers  distinct  advantages,  namely: 

1.  Absence  of  any  possible  dangerous  effects. 

2.  Absence  of  or  only  very  slight  local  and  general  reactions. 


44  ACTIVE   IMMUNIZATION 

3.  The  high  degree  and  long  duration  of  the  immunity  gained  by  pro- 
phylactic inoculations. 

4.  The  possibility  of  immunization  against  pure  parasites. 

5.  The  facility  with  which  the  inoculation  material  is  preserved. 
The  disadvantages,  however,  may  be  summarized  as  follows: 

1.  The  manufacture  of  the  inoculation  material  is  rather  complex  and 
with  some  pathogenic  bacteria  (pest),  not  without  danger. 

2.  The  increased  susceptibility  during  the  interval  between  the  inocu- 
lation and  the  onset  of  immunity. 

The  last  point  applies  not  only  to  aggressins,  but  equally  to  other 
methods  of  active  immunization.  In  times  of  an  epidemic,  aggressin 
immunization  should  never  be  undertaken. 

When  one  bears  in  mind  the  great  advantages  derived  from  the  employ- 
ment of  this  form  of  immunization,  its  extensive  use  should  be  expected; 
especially  so  as  animal  experimental  work  with  the  most  important  of  in- 
fectious bacteria:  typhoid,  cholera  (Bail),  colon  (Salus),  dysentery  (Kiku- 
chi),  staphylococcus  (Hoke),  has  proven  it  to  be  quite  successful.  It 
is  therefore  no  false  prophecy,  to  say  that  this  method  will  be  em- 
ployed more  and  more  frequently  in  the  future;  particularly  for  pest, 
results  obtained  in  animal  experimentation  by  Hueppe  and  Kikuchi  have 
more  than  sanctioned  its  employment  in  man. 

Other  methods  of  immunization -based  upon  the  Aggressin  principles 
have  been  advocated,  but  none  have  .attained  any  practical  significance. 
Mention  however  must,  in  passing,  be  made  of  the  work  of 
Brieger's  Brieger  and  his  co-workers  Mayer  and  Bassenge.  Brieger 
Bacterial  had  made  extracts  of  typhoid  and  cholera  bacilli,  in  the  main 
Extracts,  identical  with  artificial  aggressins.  As  far  as  his  sterilization 
was  concerned,  he  obtained  that  by  filtering  the  extract  through 
the  Pukal  filter.  One  should  remember  that  by  this  procedure  many 
important  substances  are  lost,  but  in  spite  of  this,  his  results  of  inoculation 
in  man  have  been  most  encouraging,  and  there  is  a  possibility  that  his 
method  may  take  the  place  of  Wright's  or  Pfeiffer  and  Kolle's,  as  the  reac- 
tions are  very  much  milder. 

Entirely  different  from  the  extracts  of  living  bacteria  are  those  made 
from  previously  killed  ones.  Neisser  and  Shiga  among  others  have  immu- 
nized against  half  parasites  in  this  manner.  This  is  not  surprising  since 
the  dead  bacterial  bodies  can  be  similarly  used  for  this  purpose.  As  a 
general  rule,  wherever  dead  bacterial  bodies  cannot  be  used  for  immuniza- 
tion, their  extracts  will  also  be  found  inefficient.  The  oldest  bacterial 
extracts  in  use  are  the  tuberculins. 


CHAPTER  V. 

TUBERCULIN  DIAGNOSIS. 

As  a  member  of  the  class  of  bacterial  extracts,  tuberculin  merits 
especial  consideration,  because  it  is  used  not  only  for  immunization,  but 
also  for  diagnostic  purposes.  Tuberculin  diagnosis  can  be  employed  in 
several  ways. 

1.  As  Koch's  subcutaneous  method. 

2.  As   the   cutaneous   reaction    (v.    Pirquet)    and    ointment  reaction 
(Moro  and  Doganoff). 

3.  As  intracutaneous  reaction. 

4.  As  ophthalmo  reaction  (Calmette). 

Koch's  Subcutaneous  Method. 

In  the  chapter  on  aggressins  it  was  shewn  that  when  a  normal  animal 
was  inoculated  with  a  certain  definite  quantity  of  bacterial  extract,  it 
could  readily  withstand  any  effects  of  such  inoculation.  If,  however,  a 
similar  quantity  was  injected  into  an  animal  previously  infected  with  the 
same  bacterium,  dangerous  symptoms  would  be  in  evidence  and  if  the  dose 
were  large  enough,  death  would  be  likely  to  follow. 

With  these  facts  for  reference,  the  following  experiments  will  be  easily 
understood.  A  number  of  tuberculous  guinea-pigs,  and  a  number  of 
normal  ones  as  control,  are  injected  with  varying  doses  of  tuberculin. 
After  twenty-four  hours  some  of  the  tuberculous  animals  are  dead,  others 
very  ill,  while  the  normal  guinea-pigs  remain  perfectly  active.  Just  as  in 
the  aggressin  experiment,  we  have  here  a  bacterial  product  in  itself  possess- 
ing only  slight  toxic  qualities  but  which  has  so  increased  the  virulence  of  the 
infection  already  existing,  that  an  ailment  which  is  usually  of  a  slowly 
progressive  nature  becomes  transformed  into  an  acute  one,  terminating 
in  the  death  of  the  animal. 

The  close  analogy  between  the  experiments  with  aggressin  as  the  in- 
jected substance,  and  that  of  the  tuberculin,  will  become  more  clear  when 
the  nature  of  the  latter  is  perfectly  understood. 

Four  to  six  weeks  old  pure  cultures  of  tubercle  bacilli  grown 

Derivation  of  in  5  per  cent,  of  glycerin  bouillon  are  filtered,  and  the  fil- 

Tuberculin.    trate  then  evaporated  down  to  i/io  of  its  original  volume. 

The  resultant  fluid,  known  as  tuberculin,  is  dark  brown  and 

syrupy  in  nature,  and  keeps  indefinitely. 

45 


46  TUBERCULIN  DIAGNOSIS 

It  consists,  therefore,  of  a  50  per  cent,  glycerin  extract  of  the  soluble  products 
of  metabolism  of  the  tubercle  bacillus. 

A  part  of  the  glycerin  has,  however,  been  used  up  for  the  nutrition  of  the  bacteria 
and  thus  it  is  highly  probable  that  after  four  to  six  weeks  the  bouillon  contains  less 
than  5  per  cent,  glycerin  and  the  evaporated  solution  less  than  50  per  cent.  The 
specific  substances  contained  within  the  tuberculin  have  not  been  definitely  established. 
As  probable  elements,  however,  may  be  recorded  products  of  secretion  of  the  living 
bacteria,  of  degeneration  of  the  dead  bacilli  and  finally  the  glycerin  soluble  substances 
extracted  from  the  bacterial  bodies  during  the  heating.  No  doubt,  all  these  substances 
and  many  others  about  which  we  lack  information,  are  directly  concerned  in  the  activ- 
ity of  the  tuberculin. 

Another  of  the  many  unsolved  questions  which  here  present  themselves  may  be 
mentioned:  whether  any  substances  exist  in  the  filtrate  which  are  thermolabile,  and 
therefore  destroyed  or  modified  by  the  heating?  According  to  Bail's  researches,  the 
aggressin  of  the  tubercle  bacillus  differs  from  all  other  aggressins  in  that  it  is  not 
thermolabile  and  can  moreover  withstand  high  grades  of  temperature.  In  spite 
of  this,  though,  attempts  to  eliminate  the  heating  during  the  manufacturing  of  the 
tuberculin  should  merit  consideration. 

If  merely  the  term  "Tuberculin"  is  used,  one  always  has  in  mind  the 
above  filtrate  tuberculin,  also  known  as  Old  Tuberculin. 

The  experiment  with  the  tuberculous  guinea-pigs  has  its  analogy  in 
the  use  of  tuberculin  in  the  case  of  man.  Here,  however,  in  order  to 
avoid  dangerous  symptoms  far  smaller  doses  of  tuberculin  are  selected. 

If  therefore  of  two  individuals  one  is  tuberculous  and  the 
The  Tuber-  other  not,  and  both  are  injected  with  the  same  amount  of  old 
culin  Reac-  tuberculin  o.ooi  c.cm.,  the  healthy  individual  remains  per- 
tioninMan.  fectly  normal  while  the  tuberculous  person  shows  a  typical 

symptom  complex  which  can  be  described  under, 

1.  General  reaction. 

2.  Focal  reaction. 

3.  Local  reaction. 

The  General  Reaction  consists  of,  fever,  headache,  malaise,  nausea, 
insomnia,  cough  irritation,  palpitation,  etc.  The  most  constant  symptom 
is  increased  temperature;  the  other  manifestations  may  only  be  very  mild 
or  even  entirely  absent. 

The  Focal  Reaction  exhibits  evidences  of  a  fresh  inflammatory  process 
in  the  suspicious  or  old  tuberculous  foci.  In  cases  of  lupus,  laryngeal,  and 
iris  tuberculosis,  this  inflammatory  reaction  can  be  distinctly  seen.  In 
pulmonary  tuberculosis  the  previously  vague  physical  signs  may  now  be- 
come definite;  rales  may  appear,  dulness  may  be  increased,  and  eventually 
pains  in  the  chest  may  arise. 

The  Local  Reaction  is  noticed  at  the  point  of  inoculation.  In  spite  of 
the  sterile  needle  and  thorough  disinfection,  the  skin  around  the  site  of 
the  injection  becomes  red,  swollen  and  painful.  That  this  is  not  due  to 
dirt  infection  is  proven  by  its  absence  in  non-tuberculous  individuals. 


HAMBURGER'S  LOCAL  REACTION  47 

[The  local  reaction  has  been  recently  advocated  by  Hamburger  as  a 
very  delicate  diagnostic  method.  He  carries  out  the  test  as  follows: 
i/io  c.cm.  of  a  i:  10,000  dilution  of  tuberculin  is  injected  just  beneath 
the  skin  of  the  forearm  or  back.  The  needle  should  be  of  fine  caliber  and 
the  syringe  should  never  have  been  used  for  more  concentrated  solutions 
of  tuberculin.  If  the  reaction  is  positive  a  subcutaneous  infiltration 
appears  within  twenty-four  hours.  Furthermore,  there  is  a  reddening 
at  the  site  where  the  point  of  the  needle  rested  (" Depot  reaction"  of 
Hamburger). 

If  there  is  no  reaction  within  twenty-four  hours,  i/io  c.cm.  of  a  dilu- 
tion i :  100  should  be  injected. 

This  subcutaneous  local  reaction  may  also  be  carried  out  in  the  form 
of  an  intracutaneous  test  (see  later)]. 

Of  the  three  types  of  reaction  the  general  and  focal  symptoms  are  the 
most  constant.  Both  are  so  characteristic  for  the  existence  of  tuberculosis, 
that  their  appearance  justifies  the  diagnosis.  In  practice,  however,  it  is 
the  general  reaction,  or  almost  exclusively  the  manifestation  of  fever,  which  is 
taken  as  the  guiding  symptom  in  Koch's  subcutaneous  method. 

The  focal  reaction  in  all  non- visible  tubercular  lesions  is  determined  by 
subjective  methods,  while  increase  in  temperature  is  alone  an  objective 
finding. 

In  carrying  out  the  subcutaneous  tuberculin  test,  one  must  remember 
several  practical  points  which  are  of  help  for  the  correct  interpretation  of 
the  results.  These  may  be  summed  up  thus: 

Inasmuch  as  the  rise  of  temperature  is  of  diagnostic  importance,  no 
patient  with  any  fever  should  be  subjected  to  the  inoculation.  For  several  days 
previous,  the  patient's  temperature  should  be  taken  every  three  hours  and 
only  if  the  temperature  does  not  exceed  37°  C.  per  axilla  should  the  tuber- 
culin diagnosis  be  undertaken. 

The  quantity  of  tuberculin  to  be  injected  is  also  of  the  utmost  consequence. 
Too  high  doses  should  be  avoided,  as  the  specificity  of  this 
osage  o  reaction,  like  all  other  biological  reactions,  is  limited  quanti- 
tatively. While  small  doses  of  tuberculin  will  give  a  rise 
of  temperature  only  in  tuberculous  individuals,  larger  doses  may  give 
the  same  rise  even  in  healthy  people.  In  addition,  too  large  doses  as  a  rule 
produce  a  general  reaction  which  might  be  very  severe  and  injurious. 

The  dosage  advised  by  Robert  Koch  for  the  diagnostic  tuberculin  reaction  is 
as  follows: 

1.  o.oooi  c.cm.  T.  (for  very  weak  individuals  and  children). 

2.  o.ooi  c.cm.  T. 

3.  0.005  c.cm.  T. 

4.  o.oi  c.cm.  T. 

5.  o.oi  c.cm.  T. 


48 


TUBERCULIN  DIAGNOSIS 


CHART  i. — Example  of  a  diag- 
nostic tuberculin  reaction. 


The  dose  chosen  at  the  first  injection  is 
o.ooi  c.cm.  T.  Very  weak  individuals,  i.e., 
those  in  an  advanced  stage  of  tuberculosis  or 
those  who  have  experienced  a  recent  hemopty- 
sis, as  well  as  children,  should  receive  an  initial 
dose  of  only  o.oooi  c.cm.  T.  Bandelier  and 
Ropke,  who  have  a  wide  experience  in  this  field, 
advise  0.0002  c.cm.  T.  as  the  primary  dose. 

Few  patients  show  a  distinctly  positive 
fever  reaction  even  with  this  small  dose;  by  a 
positive  reaction  is  meant  an  increase  in  the 
temperature  so  that  the  latter  is  at  least  0.5°  C. 
higher  than  the  highest  point  before  the  injection. 
If  the  temperature  has  not  increased,  the  re- 
action is  negative,  and  after  an  interval  of  two 
to  three  days  of  normal  temperature  the  second 
inoculation  of  0.005  c.cm.  T.  is  given.  If  as 
happens  occasionally  after  the  first  inoculation 
there  is  a  doubtful  reaction,  i.e.,  there  is  an  in- 
crease of  0.2°  to  0.3°  C.  then  the  dosage  at 
the  second  injection  should  not  be  increased 
to  0.005  c.cm.,  but  the  same  amount  o.ooi 
c.cm.  T.  is  to  be  repeated.  In  a  tuberculous 
individual  this  repeated  injection  of  o.ooi  c.cm. 
frequently  results  in  a  distinctly  positive  reac- 
tion, while  in  a  non-tuberculous  patient  instead 
of  the  former  doubtful,  a  distinct  negative  re- 
action is  obtained. 

The  general  rules  given  for  the  first  inocula- 
tion also  apply  to  the  second  with  0.005  c.cm. 
In  a  doubtful  reaction  with  this  dose,  one 
does  not  directly  proceed  to  the  o.oi  c.cm. 
dosage,  but  the  0.005  c.cm.  dose  is  repeated 
and  only  after  a  negative  reaction  with  the 
repeated  0.005  c.cm.  dose  is  the  o.oi  c.cm.  in- 
jected (see  accompanying  Chart  i).  This  rep- 
resents the  maximum  amount  of  tuberculin  to 
be  used  for  diagnostic  purposes.  Koch  advises 
repetition  of  this  dose  if  no  reaction  is  ob- 
tained. The  majority  of  authorities,  however, 
abstain  therefrom.  In  fact  some  investigators 
claim  that  a  reaction  obtained  after  inoculation 
of  o.oi  c.cm.  cannot  be  considered  specific,  be 


KOCH'S  SUBCUTANEOUS  TUBERCULIN  REACTION  49 

cause  there  are  non-tubercular  individuals  who  respond  to  this  quantity 
of  tuberculin. 

Most  tuberculous  persons  react  after  a  dose  of  o.ooi  c.cm.  to  0.005 
c.cm.  T.;  those,  however,  who  are  very  far  advanced  or  who  suffer  from 
severe  cachexia,  remain  unresponsive  to  even  much  greater  doses;  in 
addition,  patients  whose  serum  contains  antituberculin,  do  not  react  be- 
cause the  inoculated  tuberculin  is  quickly  neutralized. 

According  to  Loewenstein  the  tuberculin  reaction  does  not  depend  so 
Loewenstein's  much  upon  the  quantity  of  the  tuberculin,  as  upon  the  frequency  that 
Dosage       it  is  injected.     He,  therefore,  advises  that  the  same  amount,  about 
Scheme.       0.0002  c.cm.  be  inoculated  four  times  during  the  course  of  twelve  to 
sixteen  days.     In  by  far  the  greater  majority  of  tuberculous  patients  a 
typical  reaction  appears  after  the  third  or  fourth  injection.     The  author  has  no  per- 
sonal experience  with  this  method,  but  the  reports  of  other  authorities  do  not  exhibit 
as  favorable  results  as  those  claimed  by  Loewenstein. 

The  inoculation  is  always  to  be  given  subcutaneously,  and  the 
Technique  of  back  Qr  breast  js  the  best  site  for  it.     The  dilution  is  made 
j  .     .        immediately  before  the  injection,  with  physiological  salt  solu- 
tion or  0.5  per  cent,  carbolic  solution. 

In  interpreting  the  result  of  the  reaction  one  must  exclude 

Value  of    rises  of  temperature  due  to  extraneous  influences  such  as 

Reaction.    Angina,  Influenza,  etc.     Furthermore  there  are  individuals, 

especially  hysterical  ones,  in  whom  any  injection  as  such  is 

apt  to  produce  a  rise  of  temperature.     To  guard  against  such  a  possibility 

an  injection  cf  physiological  salt  solution  should  be  made  and  thus  quiet 

any  suspicion  of  error. 

The  diagnostic  use  of  tuberculin  is  indicated  when  one  is 
Indications,    dealing  with  adults  who  present  clinical  symptoms,  or  clinically 
suspicious    symptoms  of  tuberculosis,  but  who  run  no  tem- 
perature and  tubercle  bacilli  cannot  be  found. 
Tuberculin  is  contra-indicated  in  patients  with  high  fever, 
Contra-     and  during  or  shortly  after  hemoptysis  or  hematuria.     In 
'indications,  epilepsy,  marked  cardiac  or  renal  affection,  arteriosclerosis, 
diabetes,  and  similar  conditions,  inoculation  should  be  under- 
taken' only  under  the  strictest  indications  and  with  great  care. 

A  positive  general  reaction  means  that  the  individual  is  infected  with 
tuberculosis,  but  does  not  throw  any  light  upon  the  site,  the  extent,  or  the 
prognosis  of  the  infection.  The  focal  reaction  allows  the  diagnosis  of  the 
position  of  the  lesion. 

The  Cutaneous  Reaction. 

The  cutaneous  reaction  was  first  introduced  by  v.  Pirquet,  who  noticed 
that  by  scarification  of  the  skin  and  application  of  tuberculin,  tuberculous 


50  TUBERCULIN  DIAGNOSIS 

children  would  develop  a  distinct  papule  at  this  point,  while  in  non-tuber- 
culous conditions  such  a  reaction  would  be  absent. 

The  Technique  of  the  Cutaneous  Reaction. 

"The  patient's  forearm  on  the  inner  side  is  cleansed  with  ether;  two 
drops  of  the  pure  undiluted  old  tuberculin  are  placed  upon  the  skin  about 
10  cm.  apart,  and  then  the  skin  is  scarified  first  between  the  two  drops,  for 
the  purposes  of  a  control,  and  next  within  each  of  these  drops. — [A  boring 
scarifier,  devised  for  this,  works  very  easily.]1  Finally  a  piece  of  cotton  is 
placed  upon  each  of  these  drops  and  allowed  to  remain  there  for  ten  minutes 
after  which  the  cotton  is  removed.  A  dressing  is  not  necessary." 

Interpretations  of  the  Reaction. 

Scarification  of  itself  produces  the  so-called  "traumatic  reaction"  i.e., 
a  small  wheel  with  a  rose-colored  margin  appears  around  each  of  the  three 
points  of  scarification.  This  reaction  passes  away  after  several  hours  and 
only  a  small  scab  remains  surrounded  by  a  red  rim. 

This  "traumatic  reaction"  is  to  be  sharply  differentiated  from  the 
"specific  reaction."  The  latter  is  noticed  only  upon  the  upper  and  lower 
points  where  the  tuberculin  has  been  applied  and  consists  of  a  red,  indu- 
rated papule  which  rapidly  extends  in  size  and  elevation,  measuring  10  to  30 
mm.  in  diameter.  (Fig.  i,  Plate  I.)  The  papule  may  be  round  or  have 
irregular  margins.  Scrofulous  children  show  small,  irregularly  raised 
follicular  infiltrations  around  the  specific  reaction.  This  is  known  as  the 
"scrofulous  reaction"  It  may  appear  as  early  as  within  three  hours,  but 
usually  occurs  within  twenty-four  hours.  It  arrives  at  its  maximum 
within  forty-eight  hours;  occasionally  it  is  delayed  and  may  not  develop 
fully  until  the  third  or  fourth  day  and  then  it  begins  to  fade.  Fre- 
quently a  small  pigmented  spot  remains.  General  and  focal  reactions  are 
practically  absent. 

Moro-Doganoffs  Ointment  Reaction. 

Moro  and  DoganofT  found  that  a  50  per  cent,  ointment  of  tuberculin  in 
lanolin  rubbed  into  the  skin  without  scarification,  would  give  a  reaction 
which  consisted  of  small  nodular  or  papular  efflorescences  after  the  nature 
of  Lichen  Scrophulosorum.  In  accordance  with  the  number  and  size  of 
these  nodules  as  well  as  the  time  of  their  appearance,  three  grades  of  reaction 
are  described. 

In  employing  the  ointment  it  should  be  heated  to  25°  C.  and  a 
quantity  about  the  size  of  a  pea  is  thoroughly  rubbed  into  the  skin  of  the 

1The  flat  end  (2  mm.  wide)  of  sterile  wooden  toothpicks  can  serve  the  same  purpose. 


THE    INTRACUTANEOUS    REACTION 


abdomen  or  the  region  of  the  mammilla,  for  almost  a  minute.     The  diag- 
nostic value  of  the  reaction  is  variously  interpreted. 

An  almost  analogous  reaction,  described  independently  of  Moro,  by 
Lignieres  and»Berger  is  to  be  found  in  thoroughly  rubbing  in  concentrated 
old  tuberculin  into  the  shaved 
skin  of  tuberculous  cattle. 

The  Intracutaneous  Reaction. 

Tuberculin  even  in  very  weak 
dilutions  when  injected  directly 
into  the  skin  of  tuberculous  indi- 
viduals produces  marked  inflam- 
matory infiltrates.  This  was  first 
observed  by  Mendel  and  Man- 
toux  and  has  been  carefully 
studied  in  cattle  and  guinea-pigs 
by  Roemer. 

In  guinea-pigs  the  test  per- 
formed is  as  follows:  the  hair  of  the 
abdomen  is  removed  by  calcium 
hydrosulfid,  i/io  c.cm.  of  a  20  per 
cent,  tuberculin  solution  (0.02 
c.cm.  tuberculin)  is  injected  with 
a  very  fine  needle  directly  into  the 
skin.  The  result  is  noted  after  48 
hours  in  order  to  allow  the  trau- 
matic effects  to  wear  off.  Roemer  and  Joseph  differentiate  three  grades 
of  reaction: 

(a)  Very  susceptible  animals  (+  +  +  )  show  after  18  to  24  hours  a 
pinkish  wheal  about  the  size  of  a  half  dollar  with  a  very  deep  red  center. 
In  48  hours  the  dark  center  which  represents  a  blood  extravasation  attains 
a  greenish  hue.     After  four  days  a  superficial  necrosis  sets  in  which  leads 
to  sloughing  and  final  scarification. 

(b)  In  less  susceptible  animals  (++)  there  is  no  central  dark  area  of 
the  wheal  and  the  latter  appears  after  48  hours.     These  animals  show  a 
very  slight  necrosis  later  on. 

(c)  The  mildest  form  can  be  differentiated   from  the  traumatic  re- 
action only  in  that  it  does  not  disappear  after  48  hours  but  remains 
several  days. 

In  the  human  being  the  intracutaneous  method  has  been  advised  by 
Hamburger.  The  results  are  as  yet  insufficient  to  form  definite  conclusions 
as  to  the  clinical  value  of  the  test. 


FIG.  13. — Inoculation  with  tuberculin  for  the 
Pirquet  reaction. 


52  TUBERCULIN  DIAGNOSIS 

The  Ophthalmo  Reaction. 

At  the  discussion  which  followed  v.  Pirquet's  presentation  of  his  cuta- 
Historical.  neous  reaction,  Wolff-Eisner  remarked,  "that  by  instilling  some  10 
per  cent,  tuberculin  into  the  conjunctival  sac,  a  local  conjunctivitis  was 
obtained  and  occasionally,  also  a  general  reaction.  The  marked  severity  of  the  reac- 
tion, however,  and  its  apparent  lack  of  specificity,  made  its  diagnostic  value  improb- 
able." Calmette,  who  believed  that  Wolff-Eisner's  failure  in  obtaining  accurate 
results  lay  in  the  fact  that  glycerin  was  contained  in  the  old  tuberculin  employed  by 
him,  obtained  by  alcohol  precipitation  a  glycerin-free  dry  product,  which  he  used  in 
a  i  per  cent,  solution  equivalent  to  10  per  cent,  old  tuberculin.  It  was  he,  therefore, 
who  first  established  the  clinical  diagnostic  value  of  the  reaction.  But  his  hypothesis 
was  erroneous,  as  the  mild  reactions  which  he  obtained  were  not  due  to  the  absence  of 
glycerin,  but  because  the  Lille  tuberculin  is  much  weaker  than  the  German  prepara- 
tion. The  author  was  able  to  show  that  the  old  tuberculin  could  very  well  be  used  for 
the  Ophthalmo  reaction  if,  instead  of  the  10  per  cent.,  a  i  per  cent,  dilution  was  made. 
Thus  employed,  the  reaction  is  exceedingly  mild  and  specific.  Eppenstein  later  advised 
a  2  and  4  per  cent,  dilution  in  cases  where  the  i  per  cent,  solution  gave  no  reaction. 


Technique  of  Reaction. 

It  is  of  extreme  importance  to  have  freshly  prepared  sterile  dilutions  of 
the  old  tuberculin  (Hochst  Farbwerke).     All  the  ready-for-use  preparations 

on  the  market  should  be  discarded.  This 
applies  also  to  the  "Tuberculin  Test" 
Calmette' s  sold  by  Poulenc  Freres. 

The  mishaps  and  low  grade  of  specifi- 
city often  ascribed  in  literature  to  the 
ophthalmo  reaction  can  in  a  great  major- 
ity of  cases  be  explained  by  the  employ- 
ment of  preparations  other  than  the  i  to 
2  per  cent,  fresh  dilutions  of  the  old 
tuberculin  advocated  by  the  author,  and 
in  still  another  number  of  cases  to  its  em- 
ployment in  conditions  where  it  was  dis- 
tinctly contraindicated.  The  prepara- 

FIG.    14.— Ophthalmodiagnosticum    for  J  F     ^ 

tuberculosis.    (After  Citron.)  tion  of  fresh  tuberculin  dilutions  is  very 

much  simplified  by  the  "Ophthalmodiag- 
nosticum for  Tuberculosis ,"  of  the  firm  P.  Altmann,  Berlin  N.  W.  6  (Fig.  14). 
This  outfit  consists  of  twelve  sealed  glass  tubes  each  containing  o.i 
c.cm.  old  tuberculin,  a  cylinder  for  the  dilution  graduated  in  percentages, 
and  a  pipette  measuring  o.i  c.cm.  fitted  with  a  rubber  bulb.  One  of 
the  sealed  ampoules  is  shaken  so  that  the  tuberculin  is  collected  into  its 
broader  part  and  then  broken  at  the  designated  point  near  the  narrow  end. 
The  tuberculin  is  drawn  up  to  the  mark  into  the  pipette  and  then  trans- 


OPHTHALMO   REACTION  53 

f erred  into  the  cylinder.  Boiled  water  or  sterile  saline  is  added  to  the  i, 
2  or  4  per  cent,  dilution  mark.  The  pipette  is  washed  clean  in  the  solution 
by  successive  aspiration  and  expulsion  in  order  to  free  it  completely  of  the 
remaining  concentrated  tuberculin,  and  can  now  be  employed  as  the  eye 
dropper. 

The  solution  should  be  used  only  on  the  day  it  is  prepared.  The  tuber- 
culin in  the  sealed  tube  can  be  kept  indefinitely.  The  pipette  and  graduate 
are  sterilized  by  dry  heat,  boiling  or  by  thorough  washing  in  boiling  water. 

One  drop  of  the  tuberculin  dilution  is  deposited  in  the  inner  angle  of 
the  eye,  and  care  should  be  taken  that  the  drop  is  not  immediately  expelled, 
but  evenly  distributed  in  the  conjunctival  sac. 

In  tuberculous  individuals  the  reaction  appears  in  twelve  to 

Gradation    twenty-four  hours,   and  according  to  its  intensity   can  be 
of  the  Oph-    ,.    .  ,    ,  .    A     ^,  , 

thalmoRe-  dlvlded  mto  three  Srades' 
action.      First  Grade. — Reddening  of  the  caruncle  and  inner  side  of 

the  lower  lid  (+)  (see  Fig.  2,  Plate  I). 

Second  Grade. — Same  as  above  but  additional  involvement  of  the  con- 
junctiva of  the  eyeball  (++)• 

Third  Grade. — Conjunctivitis  purulenta,  phlyctenulae  and  other  such 
severe  manifestations  (+  +  +). 

The  reactions  of  the  first  and  second  degree  occur  most  frequently. 
The  manifestations  associated  with  the  former  of  these  are  so  mild  that  the 
patient  himself  does  not  usually  notice  them.     If  the  proper  dilution  is 
used  and  the  contraindications  of  this  test  are  observed,  a  reaction  of  the 
third  degree  is  obtained  only  in  exceptional  cases.     Fever  never  occurs. 
The  other  eye  serves  as  a  control.     It  is  advisable  therefore  before  under- 
taking the  reaction,  to  note  carefully  any  differences  that  may  exist  in  the 
conjunctive  on  both  sides.     It  must   be  remembered  that 
Selection    i-  The  greater  the  dilution,  the  more  specific  is  the  reaction, 
of  Correct    2.  The  test  should  not  be  repeated  upon  the  same  eye,  even 
Dilution,    jf  there  was  no  reaction  at  all  at  the  first  instillation. 

The  following  procedure  should  be  adopted.  A  drop  of  the 
2  per  cent,  tuberculin  dilution  is  placed  in  the  left  eye.  If  a  positive 
reaction  takes  place,  it  is  of  great  probability  that  the  patient  is  suffering 
from  an  active  tuberculous  process  and  thus  the  diagnosis  is  established. 
If,  however,  that  proves  doubtful,  and  further  corroboration  is  required, 
the  patient  should  receive,  after  the  first  reaction  has  entirely  subsided,  one 
drop  cf  a  i  per  cent,  tuberculin  dilution  in  the  right  eye. 

If  a  negative  reaction  is  obtained  at  the  instillation  of  the  2  per  cent, 
dilution,  one  drop  of  the  4  per  cent,  dilution  is  placed  in  the  right  eye.  A 
negative  reaction  with  the  4  per  cent,  mixture  speaks  almost  conclusively 
for  the  absence  of  tuberculosis  except  in  far  advanced  cachectic  condi- 
tions. A  positive  result  does  not,  on  the  other  hand,  indicate  the  presence 


54  TUBERCULIN  DIAGNOSIS 

of  tuberculosis,  as  there  are  many  normal  individuals  who  react  to  a  4  per 
cent,  tuberculin  concentration. 

Instead  of  the  tuberculin  solution,  Wolff-Eisner  recommends  a  2  per 
cent,  old  tuberculin  lanolin  ointment.  The  lower  lid  of  the  eye  is  pulled 
downward  and  a  pea- sized  mass  of  the  ointment  is  gently  placed  into  the 
conjunctival  sac  by  means  cf  a  sterile  glass  rod.  The  lid  is  held  fixed  for 
about  a  minute. 

The  ophthalmo  reaction  is  indicated  in  all  suspicious  cases  of 
Indications  tuberculosis  where  the  presence  of  bacilli  cannot  be  demon- 

, ,   ,       -r,      strated  and  where  the  subcutaneous  reaction  either  on  account 
tnalmo  Re- 
actions.    °f  the  presence  of  temperature  or  other  reasons  cannot  be 

undertaken. 

This  test  is  much  milder  and  more  agreeable  to  the  patient  than  the  sub- 
cutaneous one,  and  in  ambulatory  work  more  significant,  inasmuch  as  it 
does  away  with  any  necessity  for  considering  as  a  guide  the  temperature 
taken  by  the  untrained  and  usually  unreliable  patient. 

The  ophthalmo  reaction  is  contraindicated  in  all  diseases  of 
Contraindi-  ^e  eye^  tuberculous  or  otherwise.     If  one  eye  only  is  affected, 

ca1tl°J?s  °r  the  reaction  should  not  be  undertaken  upon  the  healthy  eye. 

the  Oph-  r,.  £     J  , 

thalmo  Re-  Similarly,  patients  who  have  had  some  eye  disease,  even  though 

action,      many  years  ago,  those  who  by  reason  of  their  occupation  are 

readily  exposed  to  eye  diseases,  or  who  live  in  districts  where 

trachoma  is  prevalent,  should  be  excluded  from  the  test.     The  reason  being 

that  in  those  individuals  the  conjunctival  mucous  membrane  becomes  a 

locus  minoris  resistentiae  and  therefore  easily  inflamed. 

Repeated  instillations  of  tuberculin  into  the  same  eye  may  set  up  very 
severe  disturbances.  Scrofulous  children  often  show  reactions  of  the  third 
degree,  inasmuch  as  they  possess  the  constitutional  tendency  which  makes 
them  easily  susceptible  to  conjunctivitis  or  phlyctenulse.  In  patients  with 
a  positive  ophthalmo  reaction  that  has  subsided,  a  recurrence  of  the  con- 
junctival inflammation  is  frequently  observed  when  they  begin  to  receive 
subcutaneous  inoculations  of  tuberculin  for  therapeutic  or  diagnostic 
purposes. 

The  Specificity  of  the  Tuberculin  Reaction. 

I'he  one  real  essential  for  the  practical  application  of  all  biological  reactions  is 
the  specificity  of  the  reaction.  There  is,  however,  as  will  be  repeatedly  pointed  out 
further  on,  no  single  absolutely  specific  reaction.  In  fact,  it  would  be  more  exact  to 
consider  these  biological  reactions  only  relatively  specific;  the  latter  depending  upon 
the  quantity  of  the  required  antigen  and  the  reacting  organism.  In  this  connection  it 
may  also  be  said,  that  it  is  never  possible  to  draw  an  exact  line  between  the  specific 
and  non-specific  biological  reactions.  There  always  will  be  a  doubtful  zone.  As  a 
general  rule,  however,  the  smaller  the  quantity  of  antigen  that  is  required  and  the 
stronger  the  resulting  reaction,  the  more  probable  is  the  biological  specificity. 


SPECIFICITY   OF  V.    PIRQUET  S   REACTION  55 

In  tuberculosis  this  problem  is  rendered  still  more  complex  by  the 
pathological  anatomical  findings,  whereby  it  is  shown  that  an  extraordinary 
high  percentage  of  individuals  have  undergone  tubercular  infection  at  some 
time  during  life.  The  clinical  consideration  of  tuberculosis,  however,  does 
not  deal  with  the  diagnosis  of  these  harmless,  practically  healed  tuberculous 
foci;  what  the  clinician  desires  to  know  is  whether  or  not  a  group  of  symp- 
toms manifested  by  a  patient  is  of  a  tuberculous  nature  or  not.  In  other 
words,  it  is  not  the  latent,  inactive,  but  the  active  form  of  tuberculosis  that 
is  to  be  diagnosed.  It  is  that  one  must  view  the  merit  of  the  various 
tuberculin  tests  from  this  standpoint. 

The  reaction  of  least  specificity  in  adults  is  the  v.  Pirquet' s  cutaneous 
reaction.  In  children  it  is  far  more  specific. 

V.  Pirquet  has  made  the  following  very  interesting  observation.. 

Out  of  747  children  in  Escherich's  clinic  in  Vienna  upon  whom  the  reac- 
tion was  tried,  there  were: 

Clinically  tuberculous  130,  out  of  which  113  (87  %)  showed  a  positive  reaction; 
Clinically  non-tuberculous  512,  out  of  which  104  (20  %)  showed  a  positive  reaction; 
Doubtful  115,  out  of  which  56  (48.6%)  showed  a  positive  reaction. 

Almost  all  of  the  tuberculous  children  who  did  not  react  were  cachectic. 

As  for  the  positive  reaction  in  non- tuberculous  cases,  the  age  of  the 
child  in  large  part  explains  the  great  differences  found. 

Whereas  healthy  infants  up  to  the  sixth  month  almost  never  give  a 
positive  reaction,  healthy  children  of 

1  to  2    years  react  in  2    per  cent,  of  cases. 

2  to  4    years  react  in  13  per  cent,  of  cases. 
4  to  6    years  react  in  1 7  per  cent,  of  cases. 
6  to  10  years  react  in  35  per  cent,  of  cases. 
10  to  14  years  react  in  55  per  cent,  of  cases. 

In  adults  one  meets  with  a  positive  v.  Pirquet's  reaction  in  more  than 
70  per  cent,  of  all  cases.  V.  Pirquet  explains  this  by  the  presence  of  latent 
tuberculosis. 

//  therefore  becomes  self-evident,  that  the  cutaneous  reaction  in  adults  is 
void  of  any  diagnostic  value.  A  negative  reaction  only,  can  be  fully  relied 
on,  and  that,  if  no  cachexia  exists. 

Ellerman  and  Erlandsen  have  attempted  to  improve  upon  the  diagnos- 
tic value  of  the  cutaneous  reaction  by  a  quantitative  titration  of  the  tu- 
berculin hypersusceptibility.  They  aimed  to  get  the  weakest  dilution 
which  still  gave  a  distinct  cutaneous  reaction.  Their  results  showed  wide 
variations.  Of  those  who  reacted  to  a  1-5  per  cent,  tuberculin  dilution  the 
great  majority  were  clinically  tuberculous;  at  the  same  time  there  were 
many  in  whom  not  the  faintest  clinical  suspicion  of  tuberculosis  could  be 
entertained. 


56  TUBERCULIN  DIAGNOSIS 

In  young  children  on  the  other  hand,  v.  Pirquet's  method  should  be 
the  one  of  choice.  In  addition  to  its  being  entirely  harmless,  and  easily 
applied,  it  possesses  a  high  diagnostic  value. 

As  for  Koch's  subcutaneous  reaction,  it  is  specific,  inasmuch  as  it  is  a  rare 
exception  to  get  a  negative  reaction  in  an  active  tuberculous  process. 
This  occurs  only  in  cases  either  with  very  severe  cachexia  or  those  with 
freely  circulating  antituberculin  in*  the  blood.  If  the  latter  two  possi- 
bilities are  excluded,  the  absence  of  a  positive  reaction  speaks  decidedly  in 
favor  of  the  absence  of  tuberculosis. 

The  interpretation  of  a  positive  reaction  as  to  the  existence  of  clinically 
active  tuberculosis  cannot  be  so  definitely  answered.  From  the  recent 
work  of  most  authorities,  however,  it  seems  to  be  taken  for  granted  that  a 
positive  reaction  does  mean  an  active  tuberculosis;  still,  this  statement 
requires  a  great  deal  of  consideration  and  limitation. 

In  this  connection  the  statistics  of  Franz  are  of  interest.  Out  of  400 
apparently  healthy  soldiers  in  one  of  the  Austrian  regiments  who  in  1901 
— their  first  year  of  service,  received  an  inoculation  of  0.003  c.cm.  of  tuber- 
culin, a  positive  result  was  found  in  61  per  cent,  of  the  cases.  In  the  fol- 
lowing year  (1902)  100  of  the  soldiers  were  re-inoculated  and  all  of  those 
who  reacted  positively  the  first  time,  did  so  a  second  time,  in  some  instances 
even  though  the  second  dosage  was  smaller.  Moreover,  fourteen  others 
who  responded  negatively  the  previous  year  showed  positive  results  this 
time,  making  a  total  of  76  per  cent.  Out  of  323  men  inoculated  for  the 
first  time  in  1902,  68  per  cent,  reacted  positively.  It  must  be  mentioned, 
however,  that  the  majority  of  the  members  of  this  regiment  came  from  a 
very  tuberculous1  district.  The  same  author  also  examined  a  Hungarian 
regiment  in  a  tuberculous-free  district,  and  under  similar  circumstances 
found  a  positive  reaction  in  38  per  cent,  of  cases.  Although  these  figures 
may  be  exceptionally  high,  they  are  without  doubt  conclusive  as  to  the 
fact  that  Koch's  reaction  cannot  be  considered  specific  for  "active" 
tuberculosis.  Franz  in  addition  gives  important  statistics  concerning  the 
health  of  the  inoculated  soldiers  whom  he  examined  for  years  following  the 
inoculation.  The  appended  charts  taken  from  the  most  recent  publica- 
tion of  Franz  (Wien.  Klin.  Woch.,  1909,  No.  28)  tabulate  what  has  been 
said  above. 


SPECIFICITY    OF    OPHTHALMO    REACTION 


57 


a 

During  the  period  of  three  years,  those 

o 

Positive 

that  terminated  their  service  through 

0> 

No.  of 

reaction 

death,  invalidity  or    long 

leave  of 

Regiment 

.£ 

soldiers 

absence  showed. 

*o 

inocu- 

§ 

> 

lated 

Negative 
reaction 

Tuberculosis 

Disease  sus- 
picious of 
tuberculosis 

Other 

diseases 

Bosn.  Inf.  Reg.  No.  i 

1901 

400 

;  +245(61%) 
\  -155(39%) 

17  (  8  deaths)             22 
5  (  4  deaths)             25 

10 

7d) 

Bosn.  Inf.  Reg.  No.  i 

1902 

323 

/  +222(68.7%) 
\  -101(31.3%) 

13  (  6  deaths' 
4 

28(1) 
13 

7(2) 
5 

Inf.  Reg.  No.  60..  .. 

1902 

279 

f+io8(38.7%) 
I  -171(61.3%) 

4 
3  (  2  deaths 

4 
>               5 

8 

12 

Total     

IOO2 

J+575 
I  -427 

34  (14  deaths 
12  (  6  deaths 

\            54(i) 
)             43 

25(2) 

24  d) 

Those    who 

reacted    in 

Regiment 

Time  of 
observation 

No.  ill  with 
manifest 
tuberculosis  i 

1901  and  1902  to  0.003 
c.cm.    tuberculin 

Positive 

Negative 

Bosn.  Inf.  Reg.  No.  i.     I  Ser.  (400 

From  10.  x.  '04 

10 

6 

4 

men). 

until    end    of 

1908. 

Same.     II    Ser.    (323    men);    Inf. 

From  Oct.,  1908, 

6 

5 

i 

Reg.  No.  60  (279  men). 

until    end    of 

1908. 

2 

i 

i 

18 

12 

6 

•         -    :'  ':  '•--'    T 

In  regard  to  the  specificity  of  the  ophthalmo  reaction,  the  condi- 
Specificity   ^QU?>  are  more  favorable  than  in  both  of  the  preceding  tuber- 
°  Reaction  Q°cu^n  tests.     The  following  short  chart  is  explanatory.     Posi- 
tive reactions  were  obtained  in 


Audeoud 

Petit 

Citron 
(ist  series) 

Eppenstein 

Schenk  and 
Seiffert 

Tuberculosis  
Suspicious  cases  

94-6% 
81.0% 

94-3% 
61.6% 

80.7% 
80.0% 

72.3% 
40.0% 

78.6% 
30.0% 

Normal  cases  .  . 

8.<% 

18.4% 

2.2% 

0.0% 

5.8% 

Calmette's  preparation. 


i%  old  tuberculin. 


58  TUBERCULIN  DIAGNOSIS 

It  is  evident  from  the  above  figures  that  by  the  use  of  the  i  per  cent,  tuber- 
culin a  grade  of  specificity  is  reached  which  can  be  considered  quite  high,  as 
the  non-tuberculous  react  only  in  a  very  small  percentage  of  cases,  while  exist- 
ing tuberculosis  is  detected  in  80  per  cent,  of  the  subjects.  Clinical  examina- 
tions of  the  positive  reacting  patients  show  that  the  latter  belong  to  the 
group  of  active  tuberculosis.  Absolute  reliance,  however,  in  the  determi- 
nation as  to  whether  the  positive  reaction  given  is  due  to  an  active  or 
latent  tuberculosis,  cannot  even  be  placed  on  the  ophthalmo  reaction. 

According  to  several  authors,  it  is  claimed  that  typhoid  fever,  rheumatism,  and 
syphilis  (in  the  stage  of  eruption)  are  very  prone  to  give  a  positive  ophthalmo  reaction, 
without  the  presence  of  a  simultaneously  existing  tuberculosis. 

In  conclusion,  therefore,  the  author  finds  it  difficult  to  make  any 
general  statement  as  to  the  preference  of  one  or  the  other  test  for  diagnostic 
purposes. 

In  children  the  application  of  the  Pirquet  reaction,  and  in  adults  the 
ophthalmo  reaction,  are  given  preference  to  Koch's  subcutaneous  reaction; 
provided,  no  contraindications  exist  against  the  first,  and  that  treat- 
ment with  tuberculin  is  not  to  be  undertaken.  In  the  latter  instance,  the 
recurrent  ophthalmo  reaction  when  the  tuberculin  therapy  is  instituted, 
authorizes  the  use  of  Koch's  subcutaneous  diagnostic  method. 

Mallein,  Trichophytin. 

Similar  to  old  tuberculin,  the  Mallein  (Helmann  and  Kelning)  has  been  obtained 
from  cultures  of  Glanders  bacilli  and  the  Trichophytin  (Plato)  has  been  isolated 
from  the  Trichophyton  fungi.  Mallein  has  already  attained  its  practical  application 
for  the  diagnosis  of  glanders  in  veterinary  medicine.  Like  tuberculin  it  is  harmless 
in  normal  organisms,  but  brings  about  temperature  and  a  local  reaction  at  the  site  of 
the  injection  when  inoculated  into  glanders  stricken  animals.  Various  general  symp- 
toms may  also  appear.  Its  employment  in  a  manner  analogous  to  the  ophthalmo 
reaction  is  also  possible. 


The  Prognostic  Value  of  the  Local  Tuberculin  Reactions. 

The  fact  that  the  tuberculin  reaction  in  cachectic  tuberculous  indi- 
viduals is  usually  negative  has  led  different  observers  to  regard  the  degree 
of  the  local  reaction  in  a  given  case  as  a  guide  to  the  prognosis.  If,  as  is 
considered,  the  strength  of  a  reaction  depends  upon  the  dose  of  tuberculin, 
the  degree  of  susceptibility  of  the  organism  and  its  reactive  power,  the 
following  theoretical  possibilities  may  be  considered. 

(a)  With  mild  clinical  manifestations,  a  reaction  obtained  only  after 
large  doses  of  tuberculin  would  speak  for  a  favorable  prognosis  because 
the  hypersusceptibility  of  the  individual  toward  tuberculin  is  still  mild. 


PROGNOSTIC  VALUE  OF  OPHTHALMO  REACTION  59 

A  severe  reaction  after  a  very  small  dose  is  less  favorable,  denoting  a  very 
much  increased  hypersusceptibility. 

(b)  With  very  marked  clinical  manifestations }  a  reaction  obtained  only 
after  large  doses  of  tuberculin  would  speak  for  an  unfavorable  prognosis 
because  the  reactive  power  of  the  organism  is  weak.  A  severe  reaction 
after  a  very  small  dose  is  more  favorable,  denoting  a  stronger  reactive 
power  of  the  individual. 

Wolf-Eisner  and  Teichmann  formulated  the  following  three  hypotheses 
in  regards  to  the  conjunctival  test. 

1.  A  positive  conjunctival  reaction  is  indicative  of  an  active  tubercu- 
losis; associated  with  manifest  clinical  symptoms  a  strongly  positive  test 
would  speak  for  a  more  favorable  prognosis  than  if  the  reaction  were  weak 
or  negative.     The  same  interpretation  can  be  applied  to  the  cutaneous 
reaction. 

2.  A  negative  reaction  (conjunctival  or   cutaneous)    with    manifest 
tuberculosis  would  point  toward  an  unfavorable  prognosis.     The  outlook 
is  also  unfavorable  if  the  ophthalmo  reaction  is  positive  but  the  cutaneous 
test  negative. 

3.  With  the  cutaneous  test,  the  local  reaction  sometimes  continues 
longer  than  four  days.     This  is  known  as  a  "  prolonged  or  continued  reac- 
tion" and  is  found  among  normal  subjects,  among  individuals  in  whom  the 
lesions  are  undergoing  a  healing  process  and  those  in  whom  the  tuber- 
culosis has  existed  for  over  ten  years.     The  prognosis  therefore  is  favorable. 


CHAPTER  VI. 
THE  TUBERCULIN  THERAPY. 

Right  at  the  beginning  it  must  be  made  clear,  that  tuberculin  is  not  to 
be  considered  as  a  curative  agent  against  tuberculosis,  but  rather  in  the 
light  of  a  bacterial  extract  for  active  immunization.  In  the  previous  chapter 
it  has  been  shown  that  while  there  are  some  infectious  diseases  where  im- 
munization can  be  accomplished  by  the  use  of  bacterial  extracts  and  dead 
bacteria,  there  are  others  where  immunization  is  possible  only  when  living 
vaccines  or  aggressins  of  living  bacteria  are  employed.  In  both  of  these 
instances,  however,  healthy  individuals  are  being  treated  to  be  protected 
from  future  infection.  An  exception  is  presented  by  rabies.  In  this  dis- 
ease, the  vaccination  against  the  active  symptoms  is  instituted  after  the 
infection  has  already  taken  place,  but  the  redeeming  feature  about  its 
treatment  is  the  existence  of  the  very  long  incubation  period.  Thera- 
peutic use  of  tuberculin,  however,  is  a  form  of  active  immunization  which 
belongs  to  neither  of  the  above  classes.  The  principle  involved  here  is 
entirely  different,  and  the  question  arises  if  it  is  at  all  possible  to  obtain  an 
active  immunity  by  the  injection  of  an  antigen  in  a  condition  where  infec- 
tion has  already  taken  place,  and  produced  pathological  changes.  [In 
other  words,  where  spontaneous  immunization  has  failed.] 

An  answer  to  this  question  is  to  be  found  in  Koch's  fundamental  ex- 
periments which  have  been  the  basis  as  well  as  starting  point  of  the  entire 
tuberculin  study. 

If  a  normal  guinea-pig  is  inoculated  with  tubercle  bacilli,  the  point  of  inoculation 
very  soon  closes.  After  ten  to  fourteen  days  there  appears  at  this  site  a  small  hard 
nodule  which  finally  ulcerates.  This  shows  no  tendency  to  heal  and  remains  so  until 
the  death  of  the  animal.  If,  however,  an  already  tuberculous  guinea-pig  is  similarly 
inoculated,  while  the  point  of  inoculation  also  closes,  no  indurated  nodule  appears. 
Instead,  a  necrotic  process  of  the  skin  sets  in  after  the  second  day,  which  finally 
terminates  in  the  casting  off  of  the  slough  and  the  formation  of  a  flat  ulceration  that 
heals  rapidly.  It  does  not  matter  whether  living  or  dead  tubercle  bacilli  are  used 
for  the  second  infection. 

In  explanation  of  the  above  phenomenon  it  must  be  said  that  although 
the  first  injection  had  a  fatal  effect  upon  the  animal,  it  must  have  stimu- 
lated certain  immune  reactions  within  the  organism  which  became  manifest 
after  the  second  inoculation.  That  a  condition  similar  to  this,  or  even 
more  favorable  exists  in  man,  is  proven  by  the  fact  that  while  the  large 

60 


IMMUNITY   AGAINST   TUBERCULIN   PREPARATIONS  6 1 

majority  of  people  become  infected  with  tuberculosis  at  some  time  during 
their  life,  only  a  small  number  show  symptoms  referable  to  the  disease  and 
the  rest  undergo  spontaneous  cure. 

Koch  further  showed  that  the  injection  of  tuberculous  guinea-pigs  with 
large  doses  of  tubercle  bacilli  produced  rapid  death,  while  frequently  re- 
peated small  doses  evinced  favorable  effects  upon  the  site  of  injection  and 
the  general  condition  of  the  animals.  In  this  way  he  proved  the  beneficial 
influence  which  successive  inoculations  exert  upon  the  primary  infection. 

In  the  employment,  however,  of  dead  tubercle  bacilli  in  man  for  thera- 
peutic purpose  a  serious  difficulty  presented  itself.  It  was  found  that  the 
inoculated  dead  bacilli  were  not  absorbed,  but  remained  for  a  long  time 
at  the  seat  of  the  inoculation  instigating  suppurative  processes.  On  intra- 
venous application,  formation  of  tubercular  nodules  was  noticed. 

Koch  realized  that  these  harmful  effects  were  due  to  the  non-absorbable 
parts  of  the  tubercle  bacilli;  in  the  main  the  bacterial  capsules.  He  there- 
fore attempted  to  extract  the  immunizing  substances,  and  in  this  way 
brought  about  the  old  tuberculin. 


It  may  be  questioned,  whether  this  old  tuberculin  is  identical  with  tuberculous 
antigen;  whether  it  is  a  feasible  preparation  for  purposes  of  immunity;  whether 
it  contains  all  the  important  elements  of  the  tubercle  bacillus;  if  not,  which  are 
lacking?  The  specificity  of  immunity  reactions  has  already  been  dealt  with 
sufficiently  to  make  it  clear  that  immunizing  a  healthy  individual  with  old  tuberculin 
will  bring  about  an  immunity  only  against  the  substances  contained  within  this  prepa- 
ration. That  that  does  not  meet  the  requirement  is  proven  by  the  fact  that  an  animal 
immunized  against  tuberculin  will  not  be  protected  against  a  later  infection  with  living 
tubercle  bacilli.  It  cannot  therefore  be  expected  that  immunization  of  a  tuberculous 
individual  with  old  tuberculin  will  protect  him  against  living  tubercle  bacilli.  The 
expectation,  however,  that  his  immunity  will  be  raised  against  old  tuberculin  only,  is 
fully  justified. 

Furthermore,  we  have  seen  in  the  aggressin  experiments,  that  inoculation  of  animals 
with  the  aggressin  antigen  was  sufficient  to  increase  the  immunity  so  that  a  subsequent 
infection  was  not  attended  by  any  harmful  effects.  In  this  case  the  injected  living 
bacteria  are  not  destroyed,  but  their  ill  effects  upon  the  immunized  organism  have  been 
paralyzed.  In  other  words,  the  parasites  have  been  transformed  to  saprophytes, 
That  a  similar  state  of  affairs  exists  in  the  use  of  antitoxic  sera  will  readily  be  seen. 
The  antitoxic  diphtheria  serum,  for  example,  neutralizes  the  toxin  and  thus  cures  the 
disease.  The  bacteria  themselves,  however,  remain  intact  and  also  infectious  for 
untreated  individuals.  Only  later  on  are  they  absorbed  by  the  phagocytes.  When 
therefore  in  an  individual  who  has  passed  through  a  course  of  tuberculin  treatment 
there  are  found  fully  virulent  tubercle  bacilli  in  the  sputum,  it  is  no  proof,  if  that  is  to 
be  the  only  corroborative  evidence,  that  the  tuberculin  treatment  had  been  ineffi- 
cient. In  fact,  there  are  strong  possibilities  that  the  tubercle  bacilli  have  become 
transformed  into  saprophytic  bacteria.  It  is,  however,  a  noteworthy  and  impor- 
tant fact,  that  immunization  with  tuberculin  proves  no  protection  against  later 
infection  with  living  tubercle  bacilli,  while  in  the  case  of  aggressins  and  toxins  this 
is  possible. 


62  TUBERCULIN  THERAPY 

Although  tuberculin  cannot  be  considered  as  the  aggressin  or  toxin  of 
the  tubercle  bacilli,  it  simulates  these  substances  with  sufficient  closeness 
to  warrant  its  use  in  tuberculosis.  It  brings  about  an  immunity  against 
some  of  the  poisonous  products  of  the  tubercle  bacillus,  leaving  the  others 
to  be  taken  care  of  by  the  natural  fighting  powers  of  the  individual. 

The  knowledge  that  this  old  tuberculin  represents  only  a  partial  aggres- 

Various       sin,  or  toxin,  and  by  that  is  meant  that  it  does  not  contain  all  the  neces- 

Tuberculin    sary  elements  for  the  establishment  of  a  true  immunity,  has  led  to  the 

Preparations,  production  of  a  large  group  of  preparations  which  are  supposed  to  supply 

the  missing  properties  of  the  old  tuberculin. 

The  more  important  of  these  preparations  were  originated  by  Robert  Koch. 
Those  which  are  of  frequent  use  are: 

a.  Old  tuberculin  (T.  Tuberculin) — preparation  described  on  page  45. 

b.  Original  old  tuberculin  (T.  O.  A.  Tuberculin  Original  Alt.) 

The  latter  consists  of  the  original  nitrate  of  the  tubercle  bouillon  culture  and  varies 
from  the  old  tuberculin  in  that  it  is  not  heated  and  reduced  to  i/io  its  volume. 
The  omission  of  heating  is  certainly  not  without  effect,  inasmuch  as  high  heat 
modifies  in  some  way  the  soluble  bacterial  substances.  This  preparation  has 
not  been  used  therapeutically  by  Koch  himself.  Spengler  and  especially  Denys,  who 
have  made  wide  use  of  it  under  the  name  of  "Le  bouillon  nitre,"  have  been  its  main 
supporters. 

c.  Vacuum  tuberculin  (V.  T.)  is  the  original  tuberculin  which  has  been  reduced  in 
vacuum  to  i/io  its  volume. 

d.  The  aqueous  tuberculin  of  Maragliano  (Tuberculina  Aquosa)  is  closely  allied  to 
the  above  tuberculins.     It  contains  all  the  water  soluble  extracts  of  the  living  tubercle 
bacilli  obtained  by  extraction  of  the  living  bacteria  in  distilled  water,  followed  by 
nitration.     As  is  evident,  it  is  prepared  on  the  same  principle  as  Brieger's  bacterial 
extracts  and  Wassermann-Citron's  artificial  aggressins. 

The  above  mentioned  tuberculin  preparations  are  all  very  much  alike 
in  that  they  contain  the  soluble  bacterial  elements.  Their  action  there- 
fore corresponds  more  or  less  to  that  of  old  tuberculin. 

Another  set  of  preparations  have  as  their  basis  the  insoluble 

New       elements  of  the  bacteria  and  cannot  as  such,  in  either  living  or 

Tuberculin   dead  form,   be   absorbed.     Since,   however,   the   absorption 

Preparations.  Of  bacteria  is  a  prerequisite  to  their  proper  action,  it  was 

necessary  to  so  alter  the  body  substances  of  these  bacteria  that 

they  could  be  taken  up.     Koch  found  that  this  was  best  accomplished  by 

thoroughly  pulverizing  the  bacilli  in  large  mortars.     And  by  this  means  the 

first  preparation  which  he  obtained  was 

e.  New  tuberculin  T.  R.  (Koch)  (Tuberculin  Rlickstand  or  Residual 
Tuberculin). 

Cultures  of  young  tubercle  bacilli  are  thoroughly  dried  in  vacuum  and  finely 
ground  in  mortars.  The  pulverized  bacilli  are  agitated  in  distilled  water  and  the 


VARIOUS    TUBERCULIN   PREPARATIONS  63 

turbid  fluid  is  centrifugalized.  The  sediment  thus  obtained  composes  the  T.  R.  or 
the  tubercle  bacilli  residue. 

T.  R.  therefore  contains  the  aqueous  insoluble  components  of  the  tubercle  bacillus, 
while  the  soluble  ones  are  retained  in  the  opalescent  supernatant  fluid  which  Koch 
calls  TO  (Tuberculin  Original). 

T.  R.  is  readily  assimilated  by  patients.  If  carefully  administered  it  produces  very 
little  infiltration  and  only  slight  temperature  and  general  reaction.  Its  price  is  com- 
paratively high  (i  c.cm.  costs  8.50  marks). 

The  first  preparation  which  contained  both  the  soluble  and  insoluble 
elements  of  the  living  bacilli  was  the 

/.  New  Tuberculin — Bacilli  emulsion  (B.  E.)  which  consists  of  T.  R.+ 
T.  O. 

The  living  tubercle  bacilli  are  first  pulverized  in  a  mortar  and  then  suspended 
in  salt  solution.  Centrifugalization  is  not  necessary,  but  sedimentation  is  required; 
50  per  cent,  glycerin  is  added  for  preservation  purposes.  Next  to  T.  the  new 
tuberculin  B.  E.  has  been  most  carefully  studied. 

B.  E.  also  lacks  in  being  an  ideal  antigen  inasmuch  as  immunity  attained  by 
its  injections  is  not  at  all  proof  against  subsequent  infection. 

Closely  resembling  the  B.  E.  is 
g.  The  Tuberculin  of  Beraneck. 

Beraneck  produced  two  tuberculin  preparations  of  which  one  is  in  the  main  identical 
with  TOA,  while  the  other  is  an  extract  of  tubercle  bacilli  with  i  per  cent,  of  phosphoric 
acid.  These  tuberculins  are  mixed  and  applied  together.  Sahli  reports  good  results 
with  this  mixture. 

Although  none  of  the  described  tuberculin  preparations  can 

Action  of     be  considered  a   true  antigen  of   the  tubercle  bacillus,  they 

Tuberculin,   undoubtedly    have    a    favorable    effect    upon    tuberculous 

individuals.     To  a  certain  extent  the  benefits  are  derived  by 

the  mechanism  of  partial  immunization.     This  in  itself  does  not,  however, 

explain  the  entire  phenomenon  of  their  successful  action. 

On  examination  of  the  tuberculous  organs  of  animals  treated  with 
tuberculin,  there  will  be  found  within  the  healthy  tissue  surrounding  the 
tuberculous  foci,  a  fresh  inflammatory  reaction.  This  consists  of  a  sero- 
fibrinous  exudate  and  a  zone  of  leucocytes  intruding  to  a  certain  extent 
upon  the  tubercular  lesion.  Tuberculin  acts  only  upon  tuberculous  tissue 
which  is  still  alive  and  not  upon  dead,  cheesy  or  necrotic  structures. 

If  enough  tuberculin  is  given  so  that  death  of  a  tuberculous  guinea-pig  occurs, 
the  changes  found  are  striking.  On  dissection,  about  the  point  of  inoculation  Koch 
reports  a  marked  congestion  of  the  blood  vessels  giving  a  red  and  often  an  almost  dark 
violet  appearance.  This  discoloration  extends  for  a  greater  or  less  distance  from  the 


64  TUBERCULIN  THERAPY 

site  in  question.  The  neighboring  lymph  glands  are  similarly  reddened.  Besides  the 
tuberculous  changes  present  within  the  liver  and  spleen,  these  organs  show  on  their 
surface  many  blackish- red  spots  varying  in  size  from  that  of  a  pin-point  to  a  hemp  seed, 
and  resembling  very  closely  the  ecchymosis  found  in  some  infectious  diseases.  On 
microscopical  examination  are  found  no  blood  extravasations,  but  very  widely  dis- 
tended capillaries  directly  surrounding  the  tuberculous  foci.  The  capillaries  are  so 
densely  plugged  with  red  blood  cells  that  it  seems  almost  impossible  for  the  circulation  to 
h  ave  continued  in  these  places.  In  exceptional  cases  only,  are  the  blood  vessels  ruptured 
and  the  escaped  blood  found  within  the  tuberculous  foci.  The  lungs  present  similar 
changes,  but  not  as  regularly  or  of  such  characteristic  appearance.  The  small  intestine 
is  often  deeply  and  evenly  congested.  In  all  this  symptom-complex,  the  never  failing 
and  almost  pathognomonic  feature  is  the  hemorrhagic-like  spots  on  the  liver  surface. 

Koch  considered  that  the  tuberculin  brought  about  the  death  of  the 
tuberculous  tissue.  He  interpreted  the  disappearance  of  the  reaction  after 
repeated  inoculations  with  tuberculin,  as  evidence  that  the  entire  tuber- 
culous structure  had  been  destroyed;  in  other  words  that  healing  had  set  in. 

Accordingly,  in  the  first  tuberculin  era,  the  erroneous  tendency  arose 
to  consider  fhose  tuberculous  patients  as  cured  who  after  gradually  dimin- 
ishing reactions  to  tuberculin  had  become  entirely  refractory  to  it. 
Actually,  these  individuals  had  merely  become  immunized  against  old 
tuberculin,  and  if  another  preparation  such  as  new  tuberculin  had  been  in- 
jected, a  reaction  would  have  recurred. 

Basing  their  conclusions  on  experimental  work,  Wassermann,  Bruck 
and  also  the  author  have  shown  that  besides  the  factor  of  partial  immuni- 
zation, it  is  the  focal  action  of  the  tuberculin  which  is  the  beneficial  agent 
in  its  therapy. 

The  inflammatory  hyperemia  produced  leads  to  a  destruction  of  the 
tuberculous  tissue,  while  at  the  same  time  the  inflammatory  process 
recedes.  In  addition  there  is  a  formation  of  connective  tissue  which  en- 
capsules  the  focus  and  with  it  also  is  associated  the  local  stimulation  of 
antibodies. 


The  Technique  of  Tuberculin-therapy. 

Three  distinct  periods  can  be  noted  in  the  history  of  this  therapy.  The  first  began 
in  the  memorable  year,  1890,  when  Robert  Koch  made  known  his  discovery  of  tuber- 
culin. At  this  time,  the  aim  of  tuberculin  treatment  was  to  cause  very  marked  reac- 
tions and  to  continue  with  the  injections  until  no  further  reaction  was  obtained.  In 
lupus,  glandular  or  bone  tuberculosis  10  mg.  was  the  initial  dose.  In  tuberculosis  of 
the  lungs  i  mg.  was  the  beginning.  If  the  patient  reacted  to  this  amount,  he  received 
daily  inoculations  of  this  dose  until  no  reaction  appeared.  Then  2  mg.  T.  were  given 
and  the  same  procedure  repeated.  Quite  frequently,  depending  upon  the  strength  of 
the  individual  concerned,  10  mg.  was  given  as  the  primary  inoculation  in  phthisis,  and 
then  rapidly  increased.  While  Koch  himself  very  soon  recognized  that  this  rather 
severe  treatment  was  suitable  only  for  incipient  or  moderately  advanced  cases,  very 
sick  and  far  advanced  phthisis  patients  were  similarly  treated  by  many  physicians. 


TREATMENT  WITH  OLD  TUBERCULIN  65 

Following  such  procedure,  decidedly  unfavorable  results  were  obtained  in  the  latter 
class  of  patients  and  consequently  a  marked  waning  in  the  enthusiasm  which  first 
greeted  the  tuberculin  therapy  was  the  inevitable  outcome.  Thus  the  once  highly 
praised  remedy  was  entirely  rejected. 

During  the  second  period  only  very  few  former  followers  of  Koch  continued  their 
studies.  They,  however,  made  it  their  business  to  investigate  the  causes  which 
accounted  for  the  failure  of  tuberculin  therapy,  Their  researches  led  to  new  prin- 
ciples in  the  treatment,  and  to  more  exact  knowledge  of  its  indications  as  well  as 
contraindications. 

The  success  obtained  by  the  untiring  efforts  of  these  investigators  brought  about 
after  many  years  a  revival  of  the  interest  in  this  therapy.  It  was  again  taken  up  (third 
tuberculin  era)  and  there  is  no  doubt  that  when  properly  handled,  tuberculin  in  well 
selected  cases  is  of  decided  benefit.  Nevertheless,  even  at  the  present  day,  a  conclusive 
opinion  as  to  many  of  its  details  cannot  be  formed.  Attempts  are  being  made  to  set 
the  individual  treatment  upon  a  biological  instead  of  upon  the  more  or  less  schematic 
basis  thus  far  employed. 

Old  Tuberculin  (Tuberculin  Koch  2Y). 

While  it  was  the  aim  in  the  early  era  of  tuberculin  treatment  to  produce 
very  strong  general  reactions,  it  is  the  general  consensus  of  opinion  at  pres- 
ent that  it  is  best  so  to  arrange  the  tuberculin  therapy  as  to  avoid  a  general 
reaction  and  especially  the  fever.  Gcetsch  was  the  man  who  first  called 
attention  to  this. 


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CHART  2. — Example  of  hyper-susceptibility  brought  about  by  going  back  to  a  smaller  dose. 

With  such  object  in  view,  one  must  begin  with  small  doses.  Some  men 
start  with  i/ioo  mg.,  others  with  i/io  mg.  T.  If  no  reaction  is  incited, 
the  dose  is  increased  in  five  to  seven  days  to  5/100  mg.  and  then  to  i/io, 
2/10, 4/10,  8/10, 1,2,4,  6,  8, 10,  20, 40,  80, 100, 150,  200, 300, 400,  600,  800, 
and  1000  mg.  This  last  amount  represents  the  maximum  dose.  If  the 
patient  still  gives  a  focal  reaction  with  such  a  dose,  it  is  best  to  repeat  it  at 
intervals  of  two  to  four  weeks,  until  finally  no  reaction  is  apparent. 
Occasionally  one  will  advance  with  the  doses  at  a  more  rapid  rate,  but  in 
general,  inoculations  should  not  be  repeated  more  than  twice  a  week. 
5 


66  TUBERCULIN  THERAPY 

If  at  any  time  a  distinct  or  even  doubtful  reaction  occurs,  it  is  absolutely 
necessary  to  await  the  complete  subsidence  of  the  latter,  and  then  the  same 
dose  is  to  be  repeated.  In  such  a  case,  an  interval  of  at  least  eight  to  ten 
days  should  elapse.  The  dosage  should  under  no  condition  be  diminished, 
as  thereby  instead  of  immunity,  hyper-susceptibility  is  the  result.  There- 
fore, patients  who  at  a  short  time  previously  had  a  diagnostic  tuberculin 
test  performed  should  receive  the  highest  dose  employed  in  this 
test  as  the  initial  prescription  for  their  tuberculin  therapy.  The 
following  chart  illustrates  the  condition  of  hyper-susceptibility  occasioned 
by  a  diminution  in  dosage.  (Chart  2.) 

Patient  had  a  localized  one-sided  apex  tuberculosis.  At  a  diagnostic  tuberculin 
injection,  he  reacted  only  when  o.oi  c.cm.  T.  was  employed.  After  the  interval  of  a 
month  the  patient  was  advised  tuberculin  treatment.  Contrary  to  the  rule  just  cited, 
he  received  as  a  first  injection  not  o.oi  tuberculin  but  0.002  c.cm.  T.  With  this  small 
dose  he  already  had  an  increase  of  temperature,  although  coming  rather  late,  and  not 
quite  typical.  After  this  reaction  had  disappeared,  without  any  other  manifestations, 
the  same  dose  of  0.002  c.cm.  tuberculin  was  repeated  and  as  evident  from  the  chart,  a 
very  marked  response  was  inaugurated.  This  was  accompanied  by  a  chill,  vomiting, 
headache,  general  pains  and  weakness.  In  addition  there  was  a  slight  relapse  after 
the  aforementioned  symptoms  had  disappeared.  In  order  to  immunize  this  patient 
against  his  hyper-susceptibility,  it  was  advisable  to  repeat  the  dose  of  0.002  c.cm.  T. ; 
the  reaction  reappeared,  but  in  a  very  much  milder  form.  It  was  only  after  the  fifth 
inoculation  of  the  same  dose  that  no  reaction  was  in  evidence.  Thus  was  the  hyper- 
susceptibility  overcome  and  the  patient  treated  in  the  general  way. 

The  danger  of  hyper-sensitiveness  also  exists  if  the  same  reactionless 
dose  is  too  frequently  repeated;  especially  so  if  the  quantities  injected  are 
small.  The  higher  the  dosage,  the  less  liable  is  the  occurrence  oj  hyper- 
susceptibility. 

This  question  is  above  all  to  be  considered  when  after  a  certain  interval, 
a  second  course  of  tuberculin  therapy  is  advised.  In  general  it  can  be  car- 
ried out  after  a  period  of  three  months,  even  though  sometimes  certain 
difficulties  may  be  met  with.  Petruschky  strongly  recommended  this 
treatment  in  successive  stages.  (Etappenbehandlung.)  The  author  is  of 
the  opinion  that  it  is  best  to  retain  the  patient  as  long  as  possible  at  his 
acquired  immunity  (tuberculin)  by  stretching  the  course  of  treatment  over 
a  long  period  of  time.  He  therefore  repeats  an  inoculation  of  the  maximum 
dose,  every  three  or  four  weeks  and  when  hyper-susceptibility  arises,  he 
changes  the  preparation  and  begins  with  a  small  dose  again. 

As  for  the  technical  details  of  the  treatment,  several  practical  sugges- 
tions may  be  made. 

1.  The  inoculation  should,  if  possible,  be  given  in  the  morning  hours, 
for  a  restless  night  usually  follows  an  injection  in  the  evening. 

2.  It  is  best  so  to  arrange  the  dilutions  that  the  patient  receives  a  frac- 
tion of  i  c.cm.  at  each  injection. 


RULES  FOR  TUBERCULIN  THERAPY 


67 


3.  The  site  of  injection  should  be  alternated  between  the  back  and  the 
breast. 

4.  The    temperature    should    be 
every   two    or    three    hours    and    a 
kept. 

5.  Disturbances  in  the  general  condition 


taken 
chart 


of  the  patient  without  the  presence  of  fever 
are  to  be  considered  in  the  light  of  general 
reactions  just  as  much  as  fever  without  other 
disturbances. 

6.  The  patient's  weight  should  be  taken 
regularly  every  week,  and  the  dose  should  be 
increased   provided   no   loss  in  weight  has 
taken  place. 

7.  In  cases  where  the  pulse  increases  in 
rate  or  becomes  poorer  in  quality,  the  treat- 
ment should  be   undertaken  very  carefully 
and  the  pulse  constantly    kept   as   guide. 
Slowness  of  pulse  can,  as  a  rule,  be  con- 
sidered a  signum  bonum. 

Especially  favorable  for  the  tuberculin 
treatment  are  the  individuals  with  a  begin- 
ning, localized  pulmonary  tuberculosis,  or 
cases  of  lupus,  and  renal  tuberculosis,  as  re- 
ported by  Lenhartz.  The  presence  of  fever 
leads  some  to  consider  such  application  as 
contraindicated.  This  is  indeed  incorrect, 
as  frequently  it  is  observed  that  a  chronic 
fever  entirely  disappears  during  a  course  of 
treatment,  and  very  often  remains  away. 
Even  if  the  fever  continues,  a  good  result  in 
the  general  condition  of  the  patient  is  never- 
theless obtained.  (Chart  3.) 


Patient  H. — Nineteen  years  old  with  distinct 
tuberculous  habitus,  on  admission  to  the  medical 
service  presented  a  marked  infiltration  and  catarrh 
of  two-thirds  of  the  right  lung  with  a  cavity  in  the 
upper  lobe;  infiltration  of  the  left  lobe  and  a  great 
number  of  tubercle  bacilli  in  the  sputum;  marked 
weakness  and  continuous  fever.  In  five  weeks  the 
patient  had  gained  n  kg.  in  weight — 8  kg.  in  one 
week. 

Simultaneously  his  general  condition  improved 
very  much;  the  night  sweats  disappeared,  and  the  cough  diminished,  but  the  number 


68  TUBERCULIN  THERAPY 

of  bacilli  still  remained  the  same,  and  the  physical  signs  of  the  lungs  unaltered.  Sub- 
sequently the  patient  received  treatment  with  B.  E.;  the  temperature  finally  sub- 
sided, the  cough  and  sputum  likewise,  and  the  bacilli  became  few  and  at  times  en- 
tirely absent  for  several  days  in  succession.  In  fact,  the  general  condition  became 
excellent.  Objectively,  there  was  no  demonstration  of  catarrhal  affection. 

It  might  be  noted  that  such  a  remarkable  increase  in  weight  in  so  short 
a  time  is  by  no  means  the  rule,  although  good  effects  are  observed  in  many 
cases. 

Naturally  the  medical  treatment  should  not  be  limited  to  the  tuberculin 
therapy.  Even  in  the  immunization  of  healthy  animals,  attention  is  paid 
to  their  housing  and  feeding;  so  much  more  imperative  is  this  consideration 
when  applied  to  sick  human  individuals. 

Bearing  in  mind  that  in  any  treatment,  success  is  only  achieved  by 
creating  a  favorable  medium,  the  same  good  influences  should  not  be 
neglected  in  tuberculin  therapy.  Rest  and  forced  feeding  are  curative 
factors  which  one  cannot  omit,  and  the  best  places  for  obtaining  these,  at 
the  beginning  at  least,  are  hospitals,  sanatoriums  or  convalescent  homes. 
When  in  such  a  way,  the  general  status  of  the  patient  is  improved,  ambu- 
lant therapy  may  be  instituted. 

As  for  the  contraindications  to  tuberculin  treatment,  it  is  very 
Contraindi-  difficult  to  set  general  rules.  Here  the  opinions  of  various 
cations  to  authorities  differ  greatly.  While  for  example,  M oiler  and 
Tuberculin  others  consider  hemoptysis  as  a  distinct  contraindication, 
Therapy.  Aufrecht  and  Kramer  claim  that  under  tuberculin  therapy 
hemoptysis  is  decidedly  improved.  It  is  easy  to  understand 
this  difference  in  attitude,  if  the  changes  in  the  focal  reaction  are  considered. 
There  is  no  doubt  that  hemoptysis  may  be  excited  by  increased  supply  of 
blood  and  the  inflammatory  process  associated  with  the  inoculation  of  tu- 
berculin. The  more  severe  the  focal  reaction,  the  greater  is  this  possibility. 
On  the  other  hand,  the  new  formation  of  connective  tissue  and  the  absorp- 
tion of  the  tuberculous  tissue  will  diminish  the  frequency  of  hemorrhage. 
With  a  general  tendency  toward  hemoptysis,  it  is  therefore  best  to  wait  a 
long  time  after  the  cessation  of  the  latter,  and  then  begin  with  small  doses. 
The  patient  should  be  under  careful  observation  and  by  constant  physical 
examination  any  possible  focal  reaction  should  be  controlled.  If,  in  spite 
of  this,  hemoptysis  does  set  in,  one  should  not  at  once  be  discouraged.  An 
interval  of  about  fourteen  days  is  to  be  allowed,  and  then  the  treat- 
ment again  undertaken.  Frequently,  the  hemoptysis  will  cease.  If  not, 
or  if  the  patient  loses  in  weight  and  becomes  weaker,  the  tuberculin  therapy 
should  be  discontinued. 

As  further  contraindications,  Moller  mentions  marked  general  weak- 
ness, fever,  heart  affections,  epilepsy,  and  hysteria.  In  full  agreement  with 
Bandelier  and  Rcepke,  the  author  does  not  consider  any  of  the  above  as 


TREATMENT  WITH  NEW  TUBERCULIN  69 

cause  for  the  non-employment  of  tuberculin.  Only  where  absolute  ca- 
chexia,  without  any  possibility  for  improvement  exists,  is  this  therapy  to  be 
omitted.  In  all  other  conditions,  an  attempt  is  by  all  means  justified. 
Experience,  of  course,  plays  an  important  r61e  in  the  selection  of  suitable 
cases.  For  a  beginner,  it  is  advisable  to  gain  practice  by  the  treatment  of 
uncomplicated  cases  before  undertaking  those  of  greater  difficulty. 

2.  New  Tuberculin-Bacilli-Emulsion  (B.  E.)  and  New  Tuberculin  T.  R. 

Treatment  with  new  tuberculin  follows  along  the  very  same  lines  set 
down  for  old  tuberculin. 

New  tuberculin  T.  R.  is  the  mildest  of  all  preparations.  It  is  very 
suitable  for  the  beginning  treatment  of  susceptible  patients.  When  the  in- 
dividual does  not  react  to  large  doses,  it  is  well  to  start  in  with  B.  E. 
B.  E.  can  also  be  employed  without  producing  any  reaction,  although  it 
is  somewhat  more  difficult. 

The  dosage  scheme  advised  by  Bandelier  and  Rcepke  is  as  follows: 

i/iooo,  2/1000,  3/1000,  7/1000,  10/1000  mg., 
15/1000,  2/100,  3/100,  5/100,  7/100,  10/100  mg., 

At  intervals  of  i  to  2  days; 
15/100,  2/10,  3/10,  5/10,  7/10,  10/10  mg., 

At  intervals  of  2  to  3  days; 
12/10,  15/10,  2,  2  1/2,  3,  mg., 
At  intervals  of  3  to  4  days; 

4,  5>  6,  7,  8,  9,  10  mg., 
at  4  to  6  to  10  days  intervals. 

In  susceptible  patients,  it  is  best  to  increase  the  dosage  only  by  one- 
half  mg.  even  when  large  doses  are  administered.  Ten  mg.  B.  E.  represents 
the  maximum  dose. 

The  author  himself  follows  a  different  scheme  from  that  of  Bandelier 
and  Rcepke.  The  injections  are  given  less  frequently,  only  about  once  a 
week,  but  the  dose  is  always  increased  twofold,  fivefold  and  even  tenfold 
without  any  excessive  reactions. 

Fever  is  obtained  much  less  often  with  new  tuberculin  than  with  T. 
The  reaction  usually  is  in  the  form  of  lassitude,  nausea,  weakness,  in- 
somnia, etc. 

The  treatment  with  new  tuberculin  is  particularly  favorable  in  cases 
where  a  low  continuous  fever  is  present.  It  also  is  more  potent  in  destroy- 
ing the  bacilli  of  the  sputum.  The  author  therefore  prefers  the  B.  E.  to 
all  other  tuberculin  preparations.  Bandelier  and  Rcepke  have  also  ob- 
tained gratifying  results  with  the  B.  E.  therapy  as  is  evident  from  the 
following  statistics  of  205  patients  treated  at  the  sanatorium  at 
Kottbus. 


TUBERCULIN  THERAPY 


Stage 

I 

II 

III 

Cured  (A)  

23  =  ii.  22% 

IO  =  37.O4% 

13  =  10.48% 

o=   o       % 

Completely  able  bodied  (BI)  

98  =  47.80% 

12  =44.44% 

77  =  62.09% 

9  =  16.63% 

Satisfactory  result  (A+BI)  

121  =  59.O2% 

22=81.48% 

90  =  72.57% 

9  =  16.63% 

Able  bodied  in  terms  of  law  (BII).  .  . 

70  =  34.15% 

5  =  18.52% 

30  =  24.19% 

35=64.81% 

Total  improvement  (A+BI-f  BII).. 
Negative  result  (C)  

191=93.17% 
I4=     6.83% 

27  =  100       % 

120  =  96.76% 
4=   3.24% 

44  =  81.44% 
10=18.56% 

Bacilli  in  sputum  on  admission  

114  =  55.61% 

4=I4.8l% 

63  =  50.81% 

47  =  87.04% 

Absence  of  bacilli  or  sputum  at  dis- 
charge. 

59  =  51.78% 

4=100     % 

49  =  77.78% 

16  =  34.34% 

The  objection  has  been  frequently  advanced  that  B.  E.  is  absorbed  with 
difficulty  and  tends  to  produce  infiltrations.  This  may  be  readily  over- 
come by  preliminary  use  of  T.  R.  or  by  the  employment  of  sensitized  B.  E., 
as  has  been  advised  by  Meyer  and  Rupple. 

By  sensitized  B.  E.  is  understood  a  bacilli  emulsion  which  has  been 
mixed  with  the  tuberculous  serum  of  a  horse  or  ox  containing  anti-tuber- 
culin. This  mixture  brings  about  a  union  between  certain  of  the  anti- 
bodies and  substances  contained  within  the  bacteria.  The  tuberculous 
serum  is  then  removed  by  centrifugalizing  and  washing  the  mixture  with 
physiological  salt  solution. 

The  sensitized  B.  E.  (S.  B.  E.)  is  milder  than  B.  E.  and  in  its  character 
is  more  like  T.  R.  The  infiltration  is  much  less,  or  entirely  absent,  due,  as 
a  general  rule,  to  the  fact  that  sensitization  of  bacteria  tends  to  neutralize 
those  substances  which  produce  infiltrations.  This  last  has  been  demon- 
strated by  the  author  in  the  case  of  mouse-typhoid,  and  swine  pest  bacilli, 
where  marked  infiltrations  following  their  inoculation  have  been  avoided 
by  sensitization.  The  following  chart  illustrates  preliminary  treatment 
with  S.  B.  E.  followed  by  B.  E.  (Chart  4.) 

The  patient  was  a  female  who  at  the  time  of  admission  presented  double-sided 
apical  and  suspicious  intestinal  tuberculosis.  Tubercle  bacilli  were  present  in  the 
sputum.  The  patient  was  discharged  from  the  clinic  as  relatively  cured,  i.e.,  all  mani- 
festations of  illness  had  disappeared  with  the  exception  of  slight  dulness  over  one  of 
the  apices  which,  however,  could  have  been  attributed  to  cicatrization.  In  addition, 
there  was  normal  vesicular  breathing,  no  temperature,  no  catarrh,  and  a  good  general 
condition.  Undoubtedly  it  is  difficult  to  say  whether  this  case  is  cured.  Only  years 
of  observation  can  prove  this.  A  temporary  latency  of  symptoms  must  always  be 
considered.  Suffice  it  to  say,  that  the  patient  was  a  great  deal  improved  and  able  to 
return  to  her  work. 


TREATMENT   WITH   SENSITIZED  BACILLI   EMULSION 


On  close  observation  of  this  chart  it  will  be  noticed  that  practically  no 
reactions  occurred  in  spite  of  the 
rather  rapid  increase  in  the  dosage. 
Slight  temperatures  were  mani- 
fest only  occasionally  (o.ooooi 
c.cm.  S.  B.  E.).  Even  though 
the  same  dose  was  not  repeated  on 
account  of  the  long  intervals 
between  the  individual  injections, 
no  increased  reaction  appeared 
after  <  subsequent  inoculation. 
When  o.i  c.cm.  of  S.  B.  E.  pro- 
duced no  reaction,  the  suscepti- 
bility to  B.  E.  was  tested.  Doses 
of  o.oooi  B.  E.  to  0.5  B.  E.  were 
administered  at  short  intervals, 
without  any  symptoms.  Only 
after  i.o  c.cm.  of  B.  E.  was  there 
a  slight  increase  of  temperature 
with  rather  marked  general  mani- 
festations— (headache,  pains  in 
the  extremities,  weakness,  etc.), 
which  subsided  within  twenty- 
four  hours.  On  repetition  of  the 
same  dose,  no  reaction  occurred. 

Six  days  later,  when  for  the 
third  time  i  c.cm.  of  B.  E.  was 
given  there  set  in  a  much  more 
marked  general  disturbance,  as 
evidence  of  hyper-susceptibility. 
(See  Chart  2.) 

3.  Bovine -tuberculin. 

Koch's  differentiation  between 
bovine  and  human  tuberculosis 
led;  to  the  attempt  at  immuniza- 
tion of  cattle  with  human  tubercle 
bacilli.  (Bovo-vaccine  of  Beh- 
ring,  and  Tauruman  of  Koch). 

Spengler  tried  to  reverse  this 
use  and  employ  the  milder,  infec- 
tious bovine  bacilli  for  the  tuberculin  therapy  in  man.  He  used  these 


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72  TUBERCULIN  THERAPY 

bacteria  to  make  up  preparations  similar  to  the  old  and  new  tuberculin. 
He  favored  especially  the  P.  T.  O.  (Perlsucht  Original  Tuberculin)  i.e., 
the  preparation  analogous  to  T.  0.  A. 

Bovine  tuberculin  is  said  to  be  borne  better  than  the  human.  The 
reactions  are  supposed  to  be  of  a  less  severe  nature,  and  the  therapeutic 
results  just  as  good  or  even  better. 

4.  Nastin. 

All  the  above-mentioned  preparations  have  as  their  aim  the  production  of  an  agent 
which  is  to  contain  the  substances  embodied  within  the  tubercle  bacilli,  and  which 
are  more  or  less  correctly  considered  as  representing  their  poisonous  elements. 
Deycke  and  Reschad  showed  that  the  fat-like  material  encapsulating  the  bacteria, 
to  which  is  ascribed  their  strongly  acid  fast  character,  also  plays  an  important 
role  in  the  question  of  tuberculosis  immunity.  These  men  prepared  a  wax-like  sub- 
stance, nastin,  from  a  strep tothrix  which  they  found  as  a  saprophyte  in  a  case  of 
leprosy — streptothrix  leproides.  Nastin  closely  resembles  the  fat-like  substance  of  the 
tubercle  bacilli  and  with  it  one  can  immunize  healthy  guinea-pigs  against  living  virulent 
tubercle  bacilli.  In  the  treatment  of  tuberculosis,  however,  it  has  no  beneficial  effect. 
On  the  day  after  inoculation,  fever  sets  in,  sputum  increases  in  great  quantities  and 
contains  large  amounts  of  tubercle  bacilli.  In  leprosy,  slight  improvement  has  been 
noticed  by  its  use. 

Metalnikoff  has  confirmed  the  above  findings  and  further  shown  that  the  bee  moth. 
Galena  Molinella,  attributes  its  very  high  immunity  against  tuberculosis  to  a  strong 
wax  dissolving  ferment  possessed  by  it.  It  is  probable,  too,  that  inoculations  of  nastin 
produce  antibodies  which  have  the  power  of  dissolving  fat.  In  this  way  the  capsule 
of  the  tubercle  bacillus  is  destroyed  and  the  antigen  is  liberated  to  be  absorbed. 
While  healthy  animals  can  thus  be  immunized,  tuberculous  individuals  would  be  indi- 
rectly receiving  a  tuberculin  injection,  and  its  amount  would  depend  upon  the  quantity 
of  tubercle  substance  suddenly  liberated.  It  seems  to  the  author  that  the  more 
rational  way  of  conducting  this  therapy  would  be,  to  first  obtain  a  high  immunity 
against  the  substance  of  the  tubercle  bacillus  by  injection  with  B.  E.,  and  then  to  follow 
this  by  treatment  with  nastin.  Such  treatment  may  prove  an  interesting  new  step  in 
tuberculin  therapy. 

Tebesapin. 

Noguchi  and  Zeuner  found  that  if  tubercle  bacilli  are  exposed  to  the  action  of 
soaps  of  unsaturated  fatty  acids,  their  capsule  is  penetrated  and  the  bacteria  are 
destroyed;  in  this  form  their  injection  into  animals  will  produce  no  infection  or  only  a 
mild  slowly  progressive  one.  Guinea-pigs  which  remain  healthy  after  receiving  such 
saponified  dead  organisms,  can  after  three  months  withstand  infection  by  virulent 
tubercle  bacilli.  At  Zeuner's  recommendation  a  preparation  known  as  Tebesapin  has 
been  put  on  the  market  by  Schering-Berlin.  It  is  made  up  in  the  following  manner: 
Tubercle  bacilli  are  shaken  for  four  days  at  a  temperature  of  37°  C.  with  an  emulsion  of 
sodium  oleate  i :  60  in  distilled  water;  the  mixture  is  then  heated  for  one  hour  at  70-72° 
C.  and  again  shaken  at  37°  C.  for  three  days;  then  centrifugalized  and  filtered.  It  is 
preserved  by  the  addition  of  0.4  per  cent,  trikresol.  Various  dilutions  of  the  prepara- 
tion can  be  obtained.  v  - 


TEBE SAFIN  73 

Preliminary  treatment  of  goats  and  guinea-pigs  with  Tebesapin  seems  to  offer  a 
certain  protection  against  future  infection.  Evidence  of  its  beneficial  action  in  man  is 
still  insufficient.  Its  harmlessness,  however,  has  been  demonstrated,  so  that  one  is 
justified  in  its  use. 

Pursuing  the  same  plan,  Deycke  and  Much  have  attempted  to  obtain  products  for 
immunization  by  shaking  tubercle  bacilli  with  lecithin,  neurin,  cholin  and  lactic  acid. 
Their  findings  have  been  disputed,  although  the  author  has  reached  conclusions  similar 
to  those  of  Deycke  and  Much.  Further  corroborative  evidence  is  necessary. 


CHAPTER  VII. 
TOXIN  AND  ANTITOXIN. 

So  far,  the  preceding  chapters  have  dealt  with  immunization  by  the 
bacterial  bodies  and  substances  extracted  from  them.  Further  attention 
must,  however,  be  paid  to  the  products  of  secretion  of  bacteria,  namely  the 
toxins.  Only  few  classes  of  bacteria  have  true  soluble  toxins  such  as  are 
possessed  by  tetanus  and  diphtheria  bacilli.  The  symptom-complex 
incited  by  the  toxin-producing  bacteria  differs  decidedly  from  that  of  the 
sepsis  class.  ,  : 

A  comparison  between  anthrax  and  tetanus  certainly  exhibits  striking 
differences.  Although  both  are  wound  infections  caused  by  characteristic 
bacteria,  smears  of  the  pus  from  wounds,  in  the  case  of  anthrax,  dis- 
play on  examination  numerous  bacilli,  while  in  the  case  of  tetanus,  the 
bacillus  is  very  sparsely  found.  Even  carefully  prepared  anerobic  cultures, 
or  inoculations  in  mice  of  the  pus  itself,  do  not  always  demonstrate  the 
tetanus  bacillus.  In  the  blood,  lymph  glands  and  viscera  of  anthrax 
cases,  excessively  large  numbers  of  microbes  can  be  found,  while  even 
in  the  most  fatal  cases  of  tetanus,  there  is  nowhere  any  evidence  of  bacteria 
or  their  spores.  Where  so  many  living  foreign  organisms  are  found  in- 
vading the  individual,  no  hypotheses  are  necessary  for  explanation  of  the 
associated  marked  disturbances  as  in  anthrax;  it  is,  however,  more 
complex  to  understand  the  severity  of  the  symptoms  in  conditions  like 
tetanus,  where  such  exceedingly  scant  bacteriological  findings  exist.  Here 
the  micro-organisms  play  only  a  secondary  role,  the  entire  symptom-com- 
plex being  produced  by  a  poison  extruded  from  the  bacteria.  In  diphthe- 
ria, conditions  are  similar  to  those  in  tetanus,  although  in  the  former  the 
bacilli  can  be  readily  demonstrated  both  microscopically  and  by  culture. 
Even  though,  however,  the  localization  of  the  bacteria  in  diphtheria  is  con- 
fined to  organs  not  absolutely  essential  for  life — diseased  tonsils — these 
themselves  do  not  explain  the  alarming  situation  observed  in  this  disease; 
the  real  cause  of  the  illness  is  to  be  found  in  the  toxin  which  is  secreted 
by  the  bacteria,  and  distributed  by  the  blood  stream  throughout  the 
entire  system. 

That  a  toxin  really  exists,  and  is  not  hypothetical,  Roux  and  Yersin,  as 
well  as  Kitasato,  have  proven  by  demonstration  of  the  poisonous  agents  in 
the  bouillon  cultures  of  both  diphtheria  and  tetanus.  As  most  cultures 

74 


ACTION   OF  DIPHTHERIA  TOXIN  75 

show  only  slight  tendencies  to  toxin  formation,  a  virulent  toxin  may  ne- 
cessitate a  special  strain  of  the  bacterium. 

The  length  of  time  required  by  cultures  for  the  production  of  moderate 
amounts  of  toxins  is  by  no  means  constant.  With  diphtheria  this  varies 
from  several  days  to  2  to  3  weeks.  As  a  general  rule,  if  toxin  is  not 
liberated  within  the  first  four  weeks  it  will  most  probably  not  appear 
after  that  time.  It  is  isolated  by  filtering  the  bouillon  culture  first 
through  filter  paper  to  remove  the  pellicle,  and  then  through  a  bacterial 
filter  to  get  rid  of  the  bacteria.  A  layer  of  toluol  i  to  2  cm.  is  added 
for  the  purposes  of  sterilization  and  it  is  advisable  to  agitate  the  toxin 
and  toluol  thoroughly  every  day  to  prevent  contamination. 

It  does  not  fall  within  the  scope  of  this  book  to  take  up  the  various 
methods  proposed  for  obtaining  and  preserving  the  various  toxins.  It  is 
the  object  merely  to  review  the  details  associated  with  their  mode  of 
action  and  standardization. 

The  first  and  most  important  member  of  this  group  is  the  diphtheria 
toxin. 

The  diphtheria  toxin  is  first  tested  by  subcutaneous  injections  into 
Action  of  guinea-pigs  250  gms.  in  weight.  The  action  of  the  toxin  is  entirely  de- 
Diphtheria  pendent  upon  the  dosage;  the  more  toxin  injected  the  more  rapidly  does 

Toxin.  death  occur.  This,  however,  is  not  to  be  taken  in  mathematically  correct 
proportions — i.e.,  twice  the  dose  does  not  produce  the  same  action  in 
one-half  the  time.  A  certain  period  of  time  must  always  elapse  before  death  can  take  place, 
the  minimum  being  about  one  day.  This  interim  is  known  as  the  period  of  incubation 
and  it  is  the  existence  of  this  that  goes  to  make  one  of  the  essential  characteristics  of  a 
true  toxin.  A  toxin  requires  a  definite  period  of  time  for  its  action  to  become  manifest; 
and  even  the  largest  dose  of  toxin  cannot  diminish  the  length  of  this  period  below  a  certain 
minimum.  On  the  other  hand,  the  length  of  the  incubation  time  can  be  increased  by 
the  injection  of  a  smaller  dose,  so  that  ultimately  a  dose  small  enough  is  obtained  which 
is  not  instrumental  in  producing  death  (Dosis  subletalis). 

If  a  guinea-pig  is  inoculated  with  a  quantity  of  toxin  sufficient  to  kill  it  in  three  to 
four  days,  nothing  abnormal  is  evident  the  first  day ;  various  manifestations  of  illness, 
however,  follow  soon  after. 

Edema  appears  at  the  site  of  inoculation.  The  animal  stops  eating,  sits  in  a  corner, 
and  reacts  poorly  to  sound.  Gradually  it  becomes  weaker,  so  that  when  placed  upon 
its  back  it  does  not  resume  its  normal  position;  the  temperature  which  at  first  rose  some- 
what, falls  abruptly  and  then  death  takes  place. 

At  autopsy,  a  gelatinous  and  strongly  hemorrhagic  edema  is  found  which  starts  at 
the  site  of  the  injection.  On  opening  the  abdominal  cavity  one  finds  but  very  little 
peritoneal  exudate,  strongly  injected  vessels  of  the  mesentery,  and  especially  charac- 
teristic, markedly  reddened  adrenal  glands.  In  the  thorax  are  found  bloody  pericar- 
dial  and  pleural  exudates,  and  consolidated  areas  in  the  lungs. 

After  the  injection  of  smaller  doses,  edema  likewise  arises  and  becomes  larger  in 
extent  the  slower  the  case  progresses.  Besides  this,  the  animal  loses  in  weight.  With 
sublethal  doses,  edema  or  infiltration  is  confined  to  the  site  of  injection,  and  finally, 
with  the  minutest  doses,  no  edema  occurs,  but  the  hair  falls  out  at  the  place  of 
injection. 


76  TOXIN  AND  ANTITOXIN 

Guinea-pigs  surviving  a  dose  of  toxin  may  after  two  to  four  weeks  begin  to  show 
paresis  first  of  the  hind,  then  of  the  fore  extremities,  and  finally  even  of  the  muscles  of 
the  back  and  respiration.  The  most  severe  types  of  such  conditions,  however,  may 
fully  subside.  They  may  be  considered  as  analogous  to  the  post-diphtheritic  paral- 
ysis taking  place  in  man,  which  is  usually  of  a  benign  nature. 

Besides  guinea-pigs  other  animals  suitable  for  diphtheria  experimental  work 
are  rabbits  (especially  by  intravenous  injection)  and  pigeons  (by  intramuscular 
injection). 

The  susceptibility  of  animals  towards  diphtheria  toxin  varies  greatly,  as  is  seen 
from  the  following  scale  of  Behring,  the  least  susceptible  animals  being  mentioned  first: 
mouse,  rat,  dog,  guinea-pig,  rabbit,  sheep,  cow,  horse,  goat. 

The  strength  of  the  diphtheria  toxin  is  estimated  as  follows: 
Estimation  Guinea-pigs  of  equal  size  (250  gms.)  receive  subcutaneous 
of  Strength  injections  of  decreasing  amounts  of  toxin.     With  a  strong 
of  Diphthe- toxin,  centi-  and  milligrams  or  even  smaller  quantities  are 
na  Toxin.  of    sufficient   potency    to   produce    death.     Doses    such    as 
these  are  not  injected  unless  diluted  in  normal  salt  solution. 
For  exact  results   one   must   not  depend  upon  the  findings  from  the 
injection  of  a  single  animal  with  each  dilution;   several  should  be  in- 
oculated with  the  same  dose  and  the  effects,  which  should  be  the  same  in 
all  cases,  noted.     It  is  impossible  to  state  beforehand  how  many  dilutions 
may  be  necessary.     If  the  various  actions  dependent  upon  the  successive 
gradations  of  dosage  are  successfully  represented,  the  experiment  may  be 
taken  as  conclusive;  that  is  to  say,  the  smallest  doses  must  leave  the  animal 
entirely  unaffected,  the  moderate  produce  slight  local  and  general  symp- 
toms, and  the  larger  ones  cause  death  of  the  animals.     If  it  should  so 
happen  that  they  all  die,  a  new  set  of  experiments  employing  a  lower 
scale  of  dosage  should  be  undertaken. 

Thus  it  is  seen  that  the  action  of  diphtheria  toxin  is  subject  to  the  quantity 
of  the  toxin  injected.  If  several  different  diphtheria  toxins  are  tested  at 
the  same  time,  it  is  at  once  evident  what  far  reaching  differences  may 
arise.  While  o.ooi  c.cm.  of  one  diphtheria  toxin  kills  a  guinea-pig  in 
twenty-four  hours,  a  different  diphtheria  toxin  will  do  the  same 
with  a  dose  ten  times  as  great,  e.g.,  o.oi  c.cm.  The  second  toxin  thus 
contains  only  one-tenth  as  many  of  the  active  substances.  In  order  to 
obtain  a  uniform  method  for  estimating  the  strength  of  a  diphtheria  toxin 
and  thus  get  comparative  values,  a  standard  unit  has  been  adopted.  And 
this  consists  of  the  smallest  amount  of  toxin  that  will  kill  a  healthy 
guinea-pig  weighing  about  250  gms.  in  four  to  five  days.  This  is  known 
as  the  minimum  lethal  dose  or  dosis  letalis  minima.  In  addition  to  this 
"direct  toxic  value/'  it  is  frequently  important,  especially  for  the  stand- 
ardization of  curative  sera,  to  estimate  the  " indirect  toxic  value" 
by  which  is  meant  the  amount  of  antitoxin  which  a  toxin  can  bind  or 
neutralize. 


DIPHTHERIA   ANTITOXIN  77 

If  an  animal,  e.g.,  a  goat  is  injected  with  a  sublethal  dose  of 
Active  Im-  diphtheria  toxin  and  after  the  lapse  of  a  certain  period  of  time 
munization  it  is  reinjected  with  a  lethal  dose,  the  animal  remains  alive, 
against     In  fact  it  may  receive  numerous  fatal  doses,  and  still  survive. 
a  Toxin.    This    experiment   is    the   simplest  in   active    immunization 
against  a  toxin.     An  examination  of  the  blood  serum  of  the 
immunized  animal  will  disclose  very  readily  what  has  taken  place.     If 
this  serum  is  mixed  with  a  fatal  dose  of  toxin  and  the  mixture  inoculated 
into  a  normal  guinea-pig,  the  latter  remains  alive  and  perfectly  active. 
The  serum  of  the  immunized  animal   therefore  contains  a 
Antitoxin,   protective  agent  which  is  directed  against  the  toxin  and  de- 
stroys its  activity;  hence  the  name  antitoxin.     But  the  anti- 
toxin is  specific,  i.e.,   diphtheria   antitoxin  neutralizes  only  diphtheria 
toxin  and  not  tetanus.     The    recognition    of    these    facts    and    those 
heretofore  mentioned,  and  the  recommendation  of  the  therapeutic  use 
of  diphtheria  serum  belong  entirely  to  v.  Behring;  righteously  may  he  be 
called  the  father  of  serum  therapy. 

Although  theoretically  the  serum  of  any  animal  immunized  with  diphtheria  toxin 
can  serve  as  a  curative  serum  for  diphtheria,  practical  experience  has  taught  that  it  is 
best  to  employ  horses  for  this  purpose.  For  laboratory  experiments  goats  should  be 
the  animals  of  choice.  It  is  advisable  to  use  the  above  animals  for  the  reason  that 
largerquantities  of  serum  are  obtained  and  furthermore  because  it  has  been  found  impos- 
sible to  immunize  guinea-pigs  with  previously  unchanged  diphtheria  toxin  even  if  the  initial 
dosage  is  the  smallest  subdivision  of  the  minimal  lethal  dose.  Behring  and  Kitashima 
showed  that  after  repeated  injections  of  very  minute  doses  they  were  able  to  kill  guinea- 
pigs  even  with  1/400  of  the  dosis  letalis  minima.  This  is  but  another  example  of  an 
effect  just  opposite  to  that  of  immunity  and  known  as  hyper  susceptibility  or  hypersensi- 
tiveness,  which  has  already  been  described  in  the  chapter  on  tuberculin  therapy.  If, 
however,  it  is  desired  to  immunize  guinea-pigs,  a  modified  form  of  the  diphtheria  toxin 
must  be  employed  for  the  first  injections.  Several  modifications  are  feasible.  Behring 
and  Kitasato  added  iodin  trichlorid  to  the  toxin  while  Roux  and  Martin  chose  Lugol's 
solution;  C.  Frankel  heated  it  to  60°  C,  and  Behring  advocated  the  so-called  "simul- 
taneous method"  (of  special  aid  in  tetanus  toxin),  where  mixtures  of  toxin  and  anti- 
toxin are  injected  and  gradually  the  quotient  of  the  latter  is  diminished  until  finally  it 
is  entirely  omitted.  If  the  animals  have  borne  the  first  inoculations  of  the  modified 
toxin  without  any  ill  effects,  one  may  then  use  the  unmodified  toxin. 

In  contrast  to  small  animals,  horses  can  be  immunized  with  unmodified  diphtheria 
toxin  right  from  the  start.  Nevertheless  great  care  must  also  here  be  exercised.  Cer- 
tain it  is,  that  less  risk  is  run  in  the  employment,  with  even  the  larger  animals,  of  a 
modified  toxin.  For  the  production  of  a  good  diphtheria  serum,  healthy  horses  about 
five  to  six  years  old  are  used  and  gradually  increasing  amounts  of  diphtheria  toxin  are 
injected  subcutaneously  or  even  intravenously;  thus  agreeing  with  Ehrlich's  findings 
to  the  effect  that  the  antitoxin  content  of  a  serum  can  be  raised  by  successively  increas- 
ing the  amount  of  toxin  injected.  As  far  as  the  efficiency  of  the  immune  serum  is 
concerned,  it  is  entirely  dependent  on  the  animal.  Horses  vary  greatly  in  their 
individual  predisposition  toward  the  production  of  an  effective  serum;  some  animals 


TOXIN  AND  ANTITOXIN 


even  completely  fail  to  do  so,  not  that  the  latter  are  not  actively  immunized,  for 
they  are,  but  because  they  contain  very  little  antitoxin  within  their  serum. 

It  is  impossible  to  recommend  a  distinct  scheme  for  the  immunization 
of  a  horse.  The  intervals  between  the  injections  and  the  size  of  the  dose 
are  varied  according  to  the  reaction  of  the  animal  toward  previous 
inoculations.  A  good  rule  to  follow  is,  that  a  fresh  injection  should  be 
given  only  if  the  reaction  from  the  preceding  one  has  entirely  subsided.  The 
reactions  are  both  local  and  general.  The  local  reaction  comes  in  the  form 
of  edema,  infiltration,  and  sterile  abscesses;  the  general,  loss  in  weight 
and  appetite  and  increase  in  temperature. 

The  following  chart  of  Salomonsen  and  Madsen,  of  the  Copenhagen  Serum  Insti- 
tute, serves  as  an  example  how  a  diphtheria  serum  is  produced.  A  gravid  mare  665  kg. 
in  weight  was  selected  and  injections  were  given  as  follows. 


Day 

Dose  of 
toxin 

Remarks 

Day 

Dose  of 
toxin 

Remarks 

i 

i  c.cm. 

I?? 

Serum  removed  contained 

6 

i  c.cm. 

oo 

150  immunity  units. 

12 

3  c.cm. 

IC4 

Birth  of  colt. 

I1? 

o  ~ 

5  c.cm. 

*OT* 

177 

Serum  removed  contained 

•••o 

23 

10  c.cm. 

/  / 

45  immunity  units. 

27 

20  c.cm. 

184 

100  c.cm. 

36 

25  c.cm. 

188 

200  c.cm. 

41 

50  c.cm. 

195 

400  c.cm. 

45 

75  c.cm. 

205 

700  c.cm. 

So 

100  c.cm. 

213 

800  c  cm. 

57 

150  c.cm. 

223 

600  c.cm. 

72 

250  c.cm. 

232 

600  c.cm. 

81 

450  c.cm. 

242 

1000  c.cm. 

02 

600  c.cm. 

2C2 

Serum  removed  contained 

y  •* 
104 

900  c.cm. 

^0  *• 

120  immunity  units. 

119 

looo  c.cm. 

- 

The  time  selected  for  venesection  is  important.  Antitoxins  like  any 
other  antibodies  do  not  arise  immediately  after  an  injection,  but  only  after 
a  certain  incubation  period.  The  amount  of  antitoxin  at  first  gradually 
increases,  then  begins  to  sink,  and  after  that  remains  constant  for  a  certain 
period  until  it  finally  disappears.  If  at  a  time  when  the  serum  contains  a 
certain  amount  of  antitoxin  a  new  inoculation  is  undertaken,  the  so-called 
"negative  phase"  sets  in,  i.e.,  the  amount  of  antitoxin  within  the  serum 
sinks.  It  is  followed  by  a  compensatory  rise,  "positive  phase."  By 
becoming  acquainted  with  the  wave-like  fluctuations  in  the  antitoxin  con- 
tent of  the  serum,  and  renewing  the  injection  at  the  time  of  highest  content, 
one  can  produce  a  serum  with  very  strong  antitoxic  qualities.  This 


STANDARDIZATION   OF   DIPHTHERIA   SERUM  79 

was  done  by  Salomonsen  and  Madsen  who  by  experimentation  found 
that  the  maximum  height  of  the  antitoxic  curve  was  reached  on  the  tenth 
day  after  each  inoculation.  For  this  reason  it  is  wise  to  choose  this  day 
for  the  removal  of  the  serum.  As  regards  other  sera,  e.g.,  tetanus,  different 
periods  have  been  empirically  found  to  be  most  serviceable.  As  the 
antitoxic  curve  does  not  remain  at  a  high  point  for  a  long  time,  the 
injections  should  be  repeated  from  time  to  time.  For  highly  immunized 
horses,  monthly  injections  usually  suffice. 

After  the  serum  has  been  obtained,  the  important  problem  which 
arises  is  how  to  keep  it  sterile.  This  is  accomplished  by  aseptic  pre- 
cautions at  the  time  of  obtaining  the  serum  and  eventually  by  the  addition 
of  preservatives  such  as  1/2  per  cent,  carbolic  acid  or  0.4  percent,  tricresol. 

This  procedure  finished,  the  next  step  is  to  estimate  the  amount  of  the 
antitoxin  content  in  the  serum. 

According  to  v.  Behring  and  Boer,  the  value  of  the  serum  should  be 
ascertained  in  respect  to  its: 

1.  Protective  power  1  .       .  r      . 

against  infection. 

2.  Curative  power      J 

3.  Protective  power  1  .     ,  .   .     .     ,. 

against  intoxication. 

4.  Curative  power      ) 

v.  Behring  found  that  these  four  properties  run  parallel  with  each  other 
so  that  for  practical  purposes,  it  suffices  to  establish  only  one  of  these 
qualities.     For  diphtheria  serum  it  has  proved  most  serviceable 
Standardiza-  to  estimate  the  strength  of  the  immunity  against  intoxica- 
tion of      tion,  since  one  is  dealing  with  a  purely  antitoxic  serum. 
Diphtheria 

Serum.       Behring's  original  mode  of  standardization  consisted  in  gradually  add- 
ing doses  of  serum  to  the  minimal  lethal  dose  of  toxin  and  injecting  the 
mixtures  into  guinea-pigs,  thus  determining  the  smallest  amount  of  serum  capable  of 
preventing  death  of  the  animal.     It  was  soon  found,  however,  that  this  method  gave 
too  inconstant  results  because  the  individual  minimal  lethal  dose  was  too  variable. 

Ehrlich,  therefore,  modified  the  process  by  using  ten  times  the  minimum  lethal  dose. 
This  amount  of  toxin,  mixed  with  decreasing  amounts  of  serum  and  made  up  to  4  c.cm. 
with  physiological  salt  solution  was  injected  subcutaneously  into  a  guinea-pig.  The 
smallest  amount  of  serum  which  saved  it  from  being  killed  on  the  fourth  to  fifth  day  was 
thus  .estimated. 

The  method  of  standardization  used  at  the  present  time  owes  its  origin 
to  Ehrlich. 

In  order  to  attain  uniformity  in  the  comparative  value  of  all  sera, 
Behring  and  Ehrlich  recommended  the  adoption  of  two  empirical  values; 
"the  normal  toxin/'  and  the  " normal  curative  serum." 

The  normal  diphtheria  toxin  is  one  which  contains  enough  toxin  in  i 
c.cm.  to  kill  25,000  gms.  of  guinea-pigs  or  100  guinea-pigs  each  weighing 
250  gms. 


80  TOXIN  AND  ANTITOXIN 

A  normal  curative  serum  is  one  of  which  o.i  c.cm.  suffices  to  neutralize 
i  c.cm.  of  Behring's  normal  poison,  i.e.,  is  able  to  overcome  the  effect  of  100 
fatal  doses,  i  c.cm.  of  this  normal  curative  serum  represents  one  immu- 
nity or  antitoxin  unit. 

The  present  antitoxin  unit  was  fixed  by  Ehrlich.  He  adopted  that 
amount  of  antitoxin  as  his  standard,  which  when  mixed  with  100  times  the 
lethal  dose  of  a  then  existing  toxin,  and  injected  into  an  animal,  was 
sufficient  to  so  neutralize  the  toxin  that  not  the  slightest  evidence  of 
either  a  local  symptom  or  general  illness  was  present.  Ehrlich  chose 
the  antitoxin  rather  than  the  toxin  as  the  constant  of  standardization, 
because  the  toxin  would  deteriorate  after  some  time,  while  the  antitoxin 
could  be  preserved  in  a  stable,  unchangeable  form. 

In  spite  of  this  fact,  the  new  method  of  titration  was  still  unsatisfactory, 
inasmuch  as  the  toxin  could  undergo  other  biological  changes  not  yet 
taken  into  account. — To  understand  these,  the  acquaintance  of  several 
new  terms  is  essential,  and  they  are,  dosis  eerie  efficax,  limes  +  or  limes 
death,  limes  o  or  limes  zero. 

While  the  dosis  letalis  minima  represents  the  smallest  dose  of  toxin 
which  may  be  fatal  in  four  to  five  days,  the  dosis  certe  efficax  (dose  of 
certain  efficiency)  stands  for  the  smallest  dose  which  will  surely  kill  any 
pig  of  250  gms.  within  this  period  of  time. 

By  limes -[-(limes  death)  is  meant  the  smallest  amount  of  toxin  which 
after  being  mixed  with  an  antitoxin  unit,  will  still  cause  the  death  of  a 
guinea-pig  within  four  to  five  days.  By  limes  o  (limes  zero)  is  understood 
the  dose  of  toxin  which  is  just  neutralized  by  one  antitoxin  unit  (I.  E.  = 
antitoxin  unit  or  "Immunitats  Einheit"),  so  that  no  toxin  is  free  and  the 
animal  remains  perfectly  well.  Limes  +  therefore  implies  an  excess  of 
poisonous  toxin;  L  0,  perfect  neutralization. 

Theoretically  speaking,  the  difference  between  L+  and  L  O  should 
represent  the  minimum  lethal  dose  (d.  1.  m.).  This,  however,  is  almost 
never  so,  as  is  shown  in  the  following  illustration. 

The  d.  1.  m.  of  a  certain  poison  was  estimated  as  0.0039  c.cm. 

L+  was  found  to  be  0.48  c.cm.  =  123  lethal  doses. 

L  0  was  found  to  be  0.42  c.cm.  =  108  lethal  doses. 


Difference  0.06  c.cm.=   15  lethal  doses. 

In  order  to  explain  this  phenomenon  Ehrlich  considered  that  there 

were  two  other  substances  contained  within  the  diphtheria  bouillon  in 

addition  to  the  diphtheria  toxin;  namely,  diphtheria  toxon  and  diphtheria 

toxoid.     The  toxon  is  a  poison  which  in  contrast  to  the  toxin 

Toxon.       has  only  a  slight  affinity  for  the  antitoxin.     It  is  this  body 

which  is  probably  the  cause  of  the  paralysis  occurring  weeks 

after  the  infection. 


STANDARDIZATION   OF   DIPHTHERIA   ANTITOXIN  8 1 

In  a  mixture  like  L  0,  the  antitoxin  has  fully  neutralized  both  the  toxon 
as  well  as  the  toxin.  If,  however,  more  diphtheria  poison  is  added  to  the 
I.  E.,  as  is  done  in  the  L  +,  the  antitoxin,  on  account  of  its  greater  attrac- 
tion for  the  toxin,  will  combine  with  the  latter  and  leave  the  toxon  free  to 
subsequently  carry  out  its  own  functions.  The  more  crude  poison  is 
added,  the  more  toxon  remains  unbound,  until  a  point  is  reached  when  no 
more  toxin  can  be  taken  up  and  consequently  some  is  leftunneutralized. 
If  the  amount  of  active  toxin  reaches  the  dosis  letalis  minima,  it  is  suffi- 
cient to  kill  the  animal  and  thus  the  limes  +  is  attained. 

When,  instead  of  freshly  prepared  toxins  Ehrlich  employed  older 
bouillon  cultures,  the  poisonous  qualities  distinctly  sank  to  about  one-half, 
but  the  surprising  fact  was  that  the  L  +  had  not  been  altered  and  even 
though  it  had  lost  one-half  of  its  toxic  power,  it  had  still  retained  its  initial 
activity  for  neutralizing  antitoxin. 

Ehrlich's  explanation  was .  that  the  diphtheria  poison  consists  of  two 
Toxoid.      molecular  groups;  one  the  carrier  of  the  toxic  qualities,  and  therefore 
known  as  the  "toxophore"  group,  the  other  uniting  with  the  antitoxin 
and  having  the  capability  of  neutralizing  it,  known  as  the  "haptophore  group."     The 
toxophore  group  is  very  labile,  while  the  haptophore  group,  strongly  in  contrast  to  it,  is 
characterized  by  its  stability.     The  toxophore  element  destroyed,  the  diphtheria 
poison  loses  its  toxic  qualities,  but  retains  its  power  to  bind  antitoxin.     A  non-poison- 
ous diphtheria  toxin  possessing  such  power  is  designated  by  Ehrlich  as  "Diphtheria 
Toxoid." 

The  mode  of  standardization  of  serum  advocated  at  the  present  day  is 
applicable  exclusively  to  the  L  +  dose.  It  is  effected  by  injecting  guinea- 
pigs  subcutaneously  with  mixtures  of  various  doses  of  diphtheria  toxin  on 
hand,  plus  an  anti-toxin  unit,  and  noting  the  smallest  amount  of  toxin 
which  kills  the  animal  in  four  to  five  days.  This  L  +  as  the  constant 
factor  is  now  mixed  with  different  amounts  of  the  serum  to  be  tested  and 
that  quantity  determined  which  just  prevents  the  death  of  the  animal. 
If  for  example  i/ioo  c.cm.  is  necessary,  this  serum  is  considered  one 
hundred  times  as  strong  as  the  standard  antitoxin  unit,  or  in  other  words 
it  contains  100  immunity  units. 

This  method  of  Ehrlich  has  been  adopted  not  only  in  Germany,  but  almost  in  all 
other  countries  in  Europe  and  also  in  America.  In  France  the  principle  varies  some- 
what, as  here  the  serum  is  tested  both  for  its  protective  and  curative  action.  The 
protective  power  of  a  serum  is  considered  50,000  if  o.oi  c.cm.  of  a  serum  saves  a  guinea- 
pig  weighing  500  gms.  from  the  fatal  consequences  following  a  dose  of  toxin  sufficient  to 
kill  an  animal  of  the  same  weight  in  thirty  to  forty  hours.  The  standard  therefore 
takes  into  consideration  the  relation  between  the  amount  of  serum  and  the  weight  of 
the  animal.  The  serum  is  injected  into  the  guinea-pig  twelve  hours  before  the  toxin 
and  the  animal  should  not  lose  in  weight  during  the  following  six  days.  The  curative 
power  is  estimated  by  injecting  a  guinea-pig  with  a  dose  of  toxin  (sufficient  to  kill  a 
6 


82  TOXIN  AND  ANTITOXIN 

control  animal  in  thirty  to  forty  hours)  and  six  hours  afterward  the  serum  is  injected. 
The  animals  remaining  alive  on  the  sixth  day  are  considered  as  cured. 

The  French  method  of  standardization  is  built  upon  the  belief  of  Roux 
that  no  parallelism  necessarily  exists  between  the  protective  and  curative 
values  of  a  serum.  Kraus  and  Schwarz  have  recently  published  accounts 
of  experiments  which  corroborate  Roux's  views.  They  claim  that  a  very 
highly  valent  diphtheria  serum  has  a  lower  curative  value  than  one  less  so; 
that  the  curative  power  of  a  serum  does  not  depend  upon  the  increase  or 
decrease  of  the  antitoxin  content  during  the  immunization  of  an  animal, 
and  that  Ehrlich's  process  of  standardization,  taking  into  consideration 
only  the  protective  power,  requires  additional  modification.  Berghaus 
working  in  Ehrlich's  Institute  answered  the  above  exceptions  in  so  satis- 
factory a  manner  that  up  to  the  present  day  Ehrlich's  views  are  still 
upheld  by  the  majority  of  workers  in  this  field. 

On  the  basis  of  former  experiments  by  Ehrlich  and  Marx,  Roemer  has 
recently  suggested  the  intracutaneous  method  for  estimating  diphtheria 
antitoxin.  Its  principle  depends  upon  the  finding  that  with  intracutaneous 
injection  of  mixtures  of  toxin  and  antitoxin,  even  the  smallest  amount  of 
toxin  if  not  fully  neutralized  will  produce  edema. 

While  in  some  countries  the  government  institutes  have  complete  control  over  the 
production  of  diphtheria  serum,  in  Germany  it  is  manufactured  by  private  concerns, 
but  under  government  supervision. 

The  serum  must  be  absolutely  clear,  free  of  bacteria  and  toxins,  especially  tetanus 
toxin,  and  must  not  contain  more  than  1/2  per  cent,  of  phenol.  It  should  contain  at 
least  the  number  of  antitoxin  units  designated  by  the  factory.. 

In  the  United  States  the  standard  antitoxin  is  distributed  by  the  Public  Health  and 
Marine  Hospital  Service  Laboratories.  Since  1902  the  production  and  sale  of  diph- 
theria antitoxin  has  been  regulated  by  law. 

At  frequent  intervals,  antitoxin  is  bought  in  the  open  market  and  examined  at  the 
hygienic  laboratories  of  the  United  States  Public  Health  and  Marine  Hospital  Service. 
Antitoxic  serum  containing  less  than  a  hundred  units  to  each  cubic  centimeter  is  pre- 
cluded from  sale. 

The  Serum  Therapy  of  Diphtheria. 

In  man  the  antitoxic  diphtheria  serum  is  used  with  success  for  both 
curative  and  prophylactic  purposes. 

For  therapeutic  application  it  is  of  the  greatest  importance  to  employ 
the  serum  in  sufficient  quantities  and  as  soon  as  possible.  The  value  of 
early  intervention  can  be  seen  from  the  following  chart  of  Kossel: 

Large  doses  of  antitoxin  should  be  administered  right  from  the  start.  The 
old  practice,  still  employed  by  few,  of  using  small  doses  is  to  be  condemned, 
for  the  aim  in  the  treatment  is  to  neutralize  as  soon  as  possible  all  the  free 
and  partly  bound  toxin. 


TREATMENT   OF   DIPHTHERIA 


Day  of  illness 

Treated 

Cured 

Died 

Percentage  of  cures 

i 

7 

7 

0 

100 

2 

7i 

69 

2 

97 

3 

30 

26 

4 

87 

4 

39 

30 

9 

77 

5 

25 

;  15 

10 

60 

6 

i?     >?• 

9 

8 

;:.:,:.   47 

7-14 

4i 

21 

20 

'•.?v;r-5^  *'••?-' 

Indefinite 

2 

I 

233 

179 

54 

77 

According  to  the  researches  of  Doenitz,  more  recently  confirmed  and 
extended  by  Fritz  Meyer,  it  was  established  that  large  amounts  of  antitoxin 
can  even  neutralize  toxin  already  attached  to  the  tissue  cells.  Men  with 
practical  experience  like  Heubner  give  4000  units  as  the  initial  dose.  In 
the  United  States  doses  as  high  as  10,000  to  100,000 1.  E.  have  been  admin- 
istered with  good  results.  The  view  of  large  dosage  is  being  gradually 
taken  up  also  in  Germany.  At  any  rate  it  is  by  far  better  to  give  too 
much  than  too  little.  If  the  first  injection  does  not  suffice  it  should  be 
repeated  the  next  day.  The  only  possible  drawback  associated  with  the 
use  of  excessive  amounts  is  the  possibility  of  serum  sickness,  to  be  men- 
tioned later.  Netter  has  found  that  the  administration  of  i  gm.  of 
calcium  chloride  on  three  successive  days  prevents  serum  sickness. 

The  serum  has  as  a  rule  been  injected  subcutaneously.  This  method 
is  very  practical  and  as  far  as  anaphylaxis  is  concerned,  is  the  least 
dangerous.  The  disadvantage,  however,  is  that  it  is  very  slowly  absorbed. 
Madsen  and  Hendersen-Smith  have  shown  that  but  a  trace  of  antitoxin 
can  be  found  in  the  blood  of  the  patient  four  and  three-fourth  hours  after 
the  injection,  and  only  after  two  to  three  days  can  larger  amounts  be 
demonstrated.  In  view  of  this,  Morgenroth  recommends  the  gluteal 
intramuscular  injection,  for  here  a  much  more  rapid  absorption  follows. 
In  cases  of  dangerous  illness  intravenous  injection  may  be  undertaken. 
For  this  purpose  Meyer  advises  a  serum  free  of  carbolic  acid,  although 
this  is  not  absolutely  necessary. 

The  importance  of  the  method  of  injection  is  clearly  shown  by  the  comparative 
experiments  of  Berghaus.  In  order  to  save  a  guinea-pig  injected  with  a  definite  amount 
of  toxin  and  followed  in  i  hour  by  antitoxin,  it  was  necessary  to  employ: 

0.08  I.  E.  by  intracardial  injection  (I.  E.  =  antitoxin  unit). 
7.0    I.  E.  by  intraperitoneal  injection. 
40.00  I.  E.  by  subcutaneous  injection. 

Thus  the  curative  power  was  increased  500  fold  by  placing  the  antitoxin  directly  into 
the  circulation. 


,84  TOXIN  AND  ANTITOXIN 

The  treatment  of  diphtheria  must  by  no  means  be  limited  to  serum 
therapy.  A  symptom  of  grave  prognosis  is  the  lowered  blood  pressure 
which  must  be  counteracted  by  infusions  of  1/2  liter  of  physiological  salt 
solution  containing  five  to  six  drops  of  adrenalin. 

The  question  whether  the  use  of  concentrated  antitoxin  is  therapeutic- 
ally  more  efficient  than  the  non-concentrated  is  still  a  matter  for  discus- 
sion. Numerous  authors  claim  that  sera  of  medium  strengths  (about 
400  I.  E.)  are  most  efficient.  The  highly  concentrated  sera  are  much 
more  expensive. 

For  prophylactic  purposes  500  to  1000  units  injected  subcutaneously 
usually  suffice.  Protection  thus  attained  lasts  about  three  weeks. 


CHAPTER  VIII. 
TOXIN  AND  ANTITOXIN  (continued). 

DEFINITION  or  TOXIN,  TETANUS  TOXIN,  BOTULISM  TOXIN,  DYSENTERY  TOXIN, 

STAPHYLOLYSIN. 

The  diphtheria  toxin  and  its  antitoxin  just  discussed  in  detail  is  of 
great  practical  and  theoretical  importance,  and  can  serve  as  a  type  of  all 
true  toxins  and  antitoxins.  Bacterial  toxins  can  be  defined  as  poisons 
given  off  by  the  bacteria,  the  symptoms  resulting  from  their  action  appearing 
after  a  certain  incubation  period.  The  invaded  organism  reacts  by  the 
production  of  specific  antitoxins  which  neutralize  the  toxins  in  amounts, 
following  the  law  of  multiple  proportions. 

Further  analysis  of  this  definition  indicates  that  a  substance  can  be 
considered  a  toxin  only  when  it  has  a  poisonous  action,  or  in  the  words  of 
Ehrlich  when  it  possesses  a  toxophore  group. 

This  toxicity  does  not  always  manifest  itself  by  necrosis  or  death  as  in 
diphtheria.  More  frequently  the  toxin  has  a  somewhat  selective  action 
affecting  a  certain  group  of  organs.  Thus  a  toxin  acting  upon  the  central 
nervous  system  or  blood  is  designated  respectively  as  a  neurotoxin  or  a 
hemotoxin.  To  differentiate  a  true  toxin  from  other  poisonous  products 
obtained  from  bacteria,  it  is  important  to  note  that  all  true  toxins  are 
elements  of  secretion  of  the  living  bacteria,  and  can  be  separated  from  them 
by  filtration.  According  to  this  definition,  poisons  contained  within  the 
bacterial  bodies  themselves,  which  may  be  liberated  by  various  mechanical, 
physical,  or  chemical  means,  cannot  be  considered  as  belonging  to  the 
class  of  true  toxins.  These  poisons  are  characterized  by  peculiar  proper- 
ties and  are  known  as  endotoxins.  In  addition  it  may  be  remarked  that 
inasmuch  as  a  true  toxin  requires  a  period  of  incubation  in  order  to 
manifest  its  action,  those  toxins  which  act  spontaneously  are  to  be  ex- 
cluded from  the  former  group.  R.  Krause  nevertheless  considered  some 
of  the  poisons  isolated  from  the  cholera  and  cholera-like  spirilla  (El  Tor 
Vibrio)  as  true  toxins  even  though  they  lack  an  incubation  period. 

The  real  essential  property  of  a  toxin  is  doubtlessly  that  one  can 
immunize  against  it,  and  be  able  to  demonstrate  the  presence  of  antitoxins 
within  the  serum  of  the  immunized  animal.  Ehrlich  claims  furthermore 
that  the  amount  of  antitoxin  produced  follows  the  law  of  multiple  propor- 
tions. By  this  is  meant  that  the  relationship  between  a  definite  dose  of 
toxin  and  the  amount  of  antitoxin  just  sufficient  to  neutralize  it,  is  constant ; 
so  that  if  ten  volumes  of  toxin  hold  in  bounds  ten  volumes  of  antitoxin, 

85 


86  TOXIN  AND  ANTITOXIN 

100  volumes  of  toxin  neutralize  100  volumes  of  antitoxin.  This  relation 
is  best  exemplified  by  the  diphtheria  toxin  and  antitoxin.  With  the  other 
toxins,  conditions  are  more  complicated  so  that  many  objections  have 
been  raised  against  the  above  rule  of  multiple  proportions.  (Bordet, 
Arrhenius,  Madsen,  etc.) 

The  true  toxins  causing  infections  in  man  are  the 

1.  Diphtheria  toxin. 

2.  Tetanus  toxin. 

3.  Botulism  toxin. 

4.  Dysentery  toxin. 

5.  Staphylolysin  and  similar  bacterial  hemotoxins. 

Tetanus  Toxin. 

The  tetanus  toxin  is  found  within  nitrates  of  bouillon  cultures 
Tetanus  of  the  tetanus  bacillus.  While  partial  erobiosis  does  not 
Toxin.  entirely  eliminate  toxin  formation,  anerobic  conditions  are  by 

far  more  favorable  for  it.  The  tetanus  toxin  is  of  two  kinds; 
the  tetanospasmin,  and  tetanolysin;  the  former  a  neurotoxin,  the  latter  a 
hemotoxin.  The  tetanospasmin  is  the  more  important  of  the  two  for  the 
reason  that  it  is  the  agent  which  produces  convulsions.  If  susceptible 
animals  such  as  mice  or  guinea-pigs  are  injected  subcutaneously  or  intra- 
muscularly with  tetanus  toxin,  after  a  certain  interval — the  incubation 
period — they  will  begin  to  show  symptoms  due  to  tetanospasmin.  They 
become  hypersensitive  to  reflex  stimulation;  clonic  convulsions  and  toxic 
rigidity  of  the  muscles  set  in.  In  animals  the  spasms  appear  first  in  the 
group  of  muscles  nearest  the  point  of  injection,  while  in  man  they 
almost  regularly  start  in  the  muscles  of  the  lower  jaw.  By  intravenous 
and  intraperitoneal  injections,  the  tetanic  spasm  appears  simultaneously 
in  all  muscles  of  the  body;  on  intracerebral  inoculation,  Roux  and  Borrel 
describe  the  occurrence  of  epileptiform  seizures,  polyuria  and  certain 
motor  disturbances — the  entire  set  of  symptoms  being  known  as  cerebral 
tetanus.  Rabbits  receiving  very  small  amounts  of  tetanus  toxin  intra- 
venously die  after  gradual  emaciation  and  marked  cachexia.  This 
type  of  infection  is  designated  by  Doenitz  as  tetanus  sine  tetano.  If 
taken  per  os,  tetanus  toxin  manifests  no  poisonous  effects.  Tetanospas- 
min is  a  distinct  nerve  poison  especially  affecting  the  central  nervous 
system. 

Experiments  by  Wassermann  and  Takaki  have  demonstrated  a  close  affinity 
existing  between  the  tetanus  toxin  and  certain  organs.  These  organs  differ  in  different 
species  of  animals.  Thus  in  man,  horse,  and  guinea-pig  only  the  central  nervous  sys- 
tem, while  in  rabbits  in  addition  to  this,  also  the  liver  and  spleen  take  up  the  tetanus 
poison.  If  an  emulsion  of  brain  tissue  and  a  fatal  dose  of  tetanus  toxin  are  mixed  and 
the  mixture  injected  into  mice,  the  latter  remain  unaffected.  According  to  Doenitz 


TETANUS   TOXIN  87 

only  the  gray  matter  and  not  the  white  substance  of  the  brain  possesses  this  absorption 
power.     If  the  brain  emulsion  is  boiled,  it  loses  this  affinity  for  the  toxin. 

Concerning  the  way  by  which  the  toxin  reaches  the  central  nervous 
system,  opinions  vary.  Most  writers,  especially  Meyer  and  Ransom, 
consider  that  the  journey  is  made  along  the  nerve  paths.  Zupnik  on 
the  other  hand  believes  that  it  is  distributed  through  the  blood  stream  and 
is  taken  up  not  only  by  the  nervous  system,  but  also  to  a  great  extent  by 
the  muscles. 

That  tetanus  toxin  is  very  labile  is  well  known.  According  to  Kitasato, 
five  minutes  at  65°  C.  or  twenty  minutes  at  60°  C.  is  sufficient  to  weaken 
the  toxicity  to  a  great  extent,  in  fact  almost  to  destroy  it.  Light  has  a 
similar  effect  upon  it.  Careful  as  its  preservation  may  be,  the  soluble 
tetanus  toxin  soon  becomes  attenuated.  Hence  the  best  way  of  keeping 
it  in  stock  is  in  a  dry  form.  For  estimating  the  strength  of  the  toxin 
white  mice  are  employed  and  are  injected  subcutaneously  with  fresh  sol- 
uble toxin,  the  lethal  dose  being  the  amount  which  kills  the  animals  in  four 
to  five  days.  Animals  more  susceptible  than  mice  are  horses,  they  being 
twelve  times  as  sensitive  and  guinea-pigs  six  times  as  much.  Hens 
possess  greater  power  of  resistance,  being  30,000  times  less  susceptible  to 
the  toxin  than  mice. 

Tetanolysin  acts  upon  the  red  blood  cells  and  disintegrates  them.  The 
erythrocytes  of  goats,  sheep  and  horses,  are  best  suited  for  experiments  to 
demonstrate  this  action.  Ehrlich  showed  that  the  tetanolysin  and  the 
tetanospasmin  are  really  two  distinctly  different  toxins  and  not  one  toxin 
with  a  twofold  function.  When  tetanus  poison  is  mixed  with  red  blood 
cells  the  tetanolysin  is  absorbed  and  the  tetanospasmin  remains  free. 
Even  the  antitoxins  of  these  two  are  different. 

As  far  as  the  standardization  of  the  tetanus  serum  is  concerned,  it 
follows  along  the  same  lines  as  the  diphtheria  serum,  i.e.,  the  L  +  dose 
of  toxin  being  the  one  employed. 

"In  America  the  method  of  standardization  was  regulated  by  a  law 
passed  in  July,  1908,  based  upon  the  work  of  Rosenau  and  Anderson  at 
the  United  States  Hygienic  Laboratories  at  Washington.  Their  unit  of 
antitoxin  is  ten  times  the  smallest  amount  of  serum  necessary  to  save  the 
life  of  a  guinea-pig  for  ninety-six  hours,  against  the  official  unit  of  standard 
toxin.  This  toxin  unit  consists  of  100  minimal  lethal  doses  of  a  precipi- 
tated toxin  preserved  at  the  hygienic  laboratory  of  the  Public  Health 
and  Marine  Hospital  Service.  At  the  hygienic  laboratory  at  Washington 
a  standard  toxin  and  antitoxin  are  preserved  under  special  conditions, 
and  standard  toxin  and  antitoxin,  arbitrary  in  their  first  establishment, 
are  kept  constant  by  being  measured  against  each  other  from  time  to 
time.  For  details  of  this  standardization  the  original  article  in  the 
United  States  Hygienic  Laboratory  Bulletin  43, 1903,  should  be  consulted." 


88  TOXIN  AND  ANTITOXIN 

Also   in  regard  to  the  efficiency  of  serum  therapy  in  tetanus 

Serum  opinions  differ.  There  is,  however,  no  doubt  that  a  certain 
Iherapy  of  .. 

Tetanus  amount  of  reliance  can  be  placed  upon  this  treatment.  Fail- 
ures in  successful  application  are  ascribed  to  the  different 
paths  by  which  the  toxin  and  antitoxin  travel.  The  former  is  carried 
by  the  nerve  fibers,  the  latter  by  the  blood  stream.  Thus  the  serum 
instead  of  being  given  subcutaneously,  as  is  the  general  rule,  is  admin- 
istered by  intraneural,  intracerebral,  and  subdural  injections.  100  to 
200  units  should  be  injected  subcutaneously  at  the  site  of  the  infection 
or  its  vicinity  and  in  addition  the  nerve  fibers  supplying  the  infected  region 
should  be  exposed  and  inoculated  with  moderate  doses  of  antitoxin  at 
various  points  along  their  centripetal  course. 

The  prophylactic  use  of  tetanus  serum  has  met  with  better  results. 
Behring  advises  the  administration  of  ten  to  twenty  antitoxin  units 
subcutaneously.  Calmette  sprinkles  upon  the  open  navel  at  birth  a 
powder  made  of  dried  serum  as  a  prophylactic  against  tetanus  neonatorum. 
Bockenheimer  advises  an  ointment  containing  the  antitoxin  as  a  dressing 
for  suspicious  wounds. 

The  Botulism  toxin  is  the  poison  produced  by  the  bacillus 

Botulism      botulinus.     This  is  the  exciting  agent  of  a  type  of  meat  and 

Toxin.        sausage  poisoning  described  by  van  Ermenghem  in  1896  as 

Botulism.     The  bacillus  botulinus  is  a  very  actively  motile 

anerobic  bacterium  which  grows   at  room    temperature   and   presents 

marked  gas  and  toxin  formation.     A  medium  in  which  the  toxin  is  readily 

produced  consists,  according  to  Ermenghem,  of  an  alkaline  bouillon  made 

in  the  form  of  an  infusion  from  ham  with  the  addition  of  i  per  cent,  of 

glucose,  i  per  cent,  of  peptone  and  i  per  cent,  of  sodium  chloride. 

The  toxin  can  be  demonstrated  after  3  weeks  of  growth,  and  is  then 
obtained  by  bacterial  filtration.  The  cultures  have  a  sour  odor  like 
butyric  acid.  The  toxin  deteriorates  easily  when  exposed  to  air  and 
light.  It  is  therefore  preserved  in  brown,  sealed  vials,  and  kept  on  ice; 
or  in  a  dried  form  in  vacuum.  Heating  the  toxin  for  three  hours  at 
58°  C.  or  one-half  hour  at  80°  C.  destroys  its  toxicity. 

Acting  unrestrained,  the  botulism  toxin  is  one  of  the  severest  of  poisons. 
It  affects  susceptible  animals  even  in  minutest  doses.  In  contradistinc- 
tion to  other  toxins  it  is  fatal  even  when  taken  per  os. 

The  characteristic  symptoms  produced  by  botulism  intoxication  consist  of  hyper- 
secretion  of  mucus  from  the  mouth  and  nose,  paralysis  of  eye  muscles,  urine  retention, 
obstipation,  dysphagia,  aphagia,  and  aphoria.  No  fever,  nor  any  sensitory  disturb- 
ances are  in  evidence.  Death  takes  place  because  of  bulbar  paralysis  accompanied  by 
respiratory  and  cardiac  failure, 

The  poison  is  absorbed  or  arrested  in  the  central  nervous  system,     o.i 


BOTULISM   TOXIN  89 

c.cm.  of  an  emulsion  of  central  nervous  tissue  neutralizes  three  times  the 
fatal  dose  for  mice.  Lecithin,  cholesterin,  as  well  as  fatty  substances 
like  butter  and  oil,  act  in  a  similar  manner. 


Monkeys,  rabbits,  guinea-pigs,  mice  and  cats  are  susceptible  to  the  toxin. 

Cats  usually  exhibit  the  most  characteristic  clinical  picture.  Localized  and  almost 
pathognomonic  paralyses  occur  in  the  form  of  prolapse  of  tongue,  marked  mydriasis, 
aphonia,  aphagia,  etc. 

In  mice,  paralysis  of  the  hind  extremities  sets  in  after  quite  a  small  dose;  and  death 
follows  in  a  few  hours. 

In  rabbits  and  guinea-pigs,  moderate  doses  (0.0003-0.001  c.cm.)  occasion  no  mani- 
festations during  the  first  two  to  three  days,  but  subsequently,  the  above-mentioned 
paralyses  arise  and  in  several  hours  the  animals  expire.  With  larger  doses  (o.i  to 
0.5  c.cm.)  the  incubation  period  lasts  only  a  couple  of  hours  and  then  dyspneic  attacks 
usually  succeeded  by  motor  paralysis  and  death  are  the  consequences. 

The  strength  of  the  botulism  toxin  is  ascertained  by  injecting  guinea-pigs  subcu- 
taneously  and  observing  the  time  when  loss  in  weight,  flabbiness  of  abdominal  muscles 
and  death  occur. 

The  following  chart  by  Madsen  exhibits  the  above  principle.    (+  means  "death.") 


Dose  in  c.cm. 

Result 

Dose  in  c.cm. 

Result 

cxoois 

+on  i  st.  day 

0.0009 

Weakness  in  3  weeks. 

0.0015                  +  after  11/2  days 

0.0009 

Loss  in  weight. 

0.0015 

+  after  2         days 

0.0009 

Loss  in  weight. 

0.0013                  +  after  2         days 

0.0007 

Loss  in  weight  in  2  weeks. 

0.0013                  +  after  5         days 

0.0007 

Loss  in  weight  in  i  week. 

0.0013 

+  after  6         days 

0.0007 

Loss  in  weight  in  i  week. 

O.OOI 

+  after  4         days 

0.0005 

Loss  in  weight  in  i  week. 

o.oo  i                   +  after  5         days 

0.0003 

Practically  no  symptoms;  only 

O.OOI 

-f  after  51/2  days 

several  days  of  weakness. 

Kempner  immunized  goats  against  botulism  toxin  and  proved  the  presence  of  anti- 
toxins within  their  sera.  Immunization  of  rabbits  and  guinea-pigs  is  only  feasible  if 
primary  inoculations  are  made  with  a  toxin  previously  attenuated  by  heat  for  one- 
half  hour  at  60°  C. 

Recently  Wassermann  has  immunized  horses  against  this  toxin.  In 
mew  of  the  high  mortality  and  lack  of  any  other  specific  medication,  the  use  of 
this  serum  is  strongly  advised.  In  animal  experimentation  it  shows  itself 
of  undeniable  value.  As  for  its  effects  in  man,  it  has  not  been  employed 
frequently  enough  to  judge. 

The  botulism  toxin  and  antitoxin  unite  only  very  slowly.  Otto  and  Sachs  have 
shown  that  the  inoculation  of  rabbits  with  a  three  hours  old  mixture  of  toxin  and  anti- 
toxin occasioned  greater  toxic  effects  when  administered  intravenously  than  when  given 
subcutaneously.  Only  in  mixtures  twenty-four  hours  old  was  this  difference  overcome. 


pO  TOXIN  AND  ANTITOXIN 

Dysentery  toxin  was  first  demonstrated  by  Conradi.     Subse- 

Dysentery  quently  from  experiments   by  Rosenthal,  Todd,  Kraus   and 

Toxin.      Doerr,  etc.,  it  became  evident  that  this  was  a  true  toxin  and  not 

an  endo toxin  as  was  originally  considered.  Only  the  Kruse- 
Shiga  type  of  bacillus  forms  a  toxin;  for  the  Flexner  type,  no  definite  toxin 
has  as  yet  been  isolated.  Recent  investigators,  however,  especially  Kraus 
and  Doerr,  are  inclined  to  consider  the  human  dysentery  of  the  Kruse- 
Shiga  origin  in  the  light  of  an  intoxication  or  toxemia  similar  to  diphtheria. 
The  lesions  in  the  large  intestine  where  the  bacteria  accumulate  can  be 
compared  to  the  diseased  diphtheria  tonsils,  while  the  other  manifestations, 
as  the  cerebral  symptoms,  cardiac  disturbances,  nervous  sequelae,  eye 
affections,  etc.,  can  be  taken  as  expressions  of  the  toxemia. 

Like  the  other  described  toxins,  the  dysentery  toxin  can  be  obtained  by  nitration  of 
bouillon  cultures.  The  meat  infusion  must  be  quite  alkaline.  The  optimum  alkalin- 
ity, according  to  Doerr,  is  obtained  by  adding  0.3  per  cent,  soda  to  litmus  neutral 
bouillon.  The  precipitate  thus  formed  which  increases  on  sterilization  should  not  be 
removed  by  nitration.  Doerr  also  advises  finely  powdered  chalk  (20  gms.  pro  liter)  to 
be  added  to  the  weakly  alkaline  bouillon  before  the  last  sterilization.  The  toxin  is 
formed  very  gradually;  the  maximum  is  derived  after  two  to  three  weeks.  The  gray 
white  pellicle  upon  the  surface  of  the  culture  can  be  taken  as  an  indicator  for  the  amount 
of  toxin  present. 

According  to  Kraus  a  good  dysentery  toxin  can  also  be  made  by  emulsifying  the 
bacteria  (grown  upon  agar)  in  physiological  salt  solution  and  filtering  through 
Reichel  niters. 

The  toxicity  of  individual  strains  of  dysentery  bacilli  varies  greatly. 

The  strength  of  the  toxin  is  diminished  by  heating  for  one  to  two  hours  at  60°  C. 
Higher  temperatures  destroy  it:  80°  C.,  in  three  minutes  and  90°  to  100°  C.  in  one 
minute. 

Acids  destroy  the  toxin  probably  by  the  formation  of  a  non-poisonous  compound. 
The  addition  of  a  strong  alkali  restores  the  toxicity. 

Its  preservation  can  be  accomplished  in  a  fluid  state  under  the  cover  of  toluol. 

The  action  of  dysentery  toxin  can  best  be  studied  by  its  effect  upon 
rabbits  after  intravenous  inoculation.  Large  doses  kill  the  animals  in 
very  short  time,  six  to  seven  hours.  The  ordinary  lethal  dose  produces 
characteristic  symptoms  consisting  of  paresis,  diarrhea,  which  may  be 
bloody,  paralysis  of  the  bladder,  hypothermia,  etc.  Death  takes  place  in 
three  to  four  weeks. 

Given  subcutaneously,  or  intraperitoneally,  the  toxin  has  only  a  very 
mild  action.  The  incubation  period  is  especially  prolonged.  Given  per 
os,  no  effect  is  in  evidence. 

Besides  rabbits  the  other  susceptible  animals  (to  large  doses)  are  monkeys,  cats 
and  dogs;  chickens,  pigeons  and  guinea-pigs  are,  in  the  opinion  of  Kraus  and  Doerr, 
not  at  all  affected  by  the  toxin. 


DYSENTERY   SERUM  9 1 

The  intestinal  changes  found  at  post  mortem  examination  of  the  animals  very 
closely  simulate  the  pathological  alterations  occurring  in  man.  A  hemorrhagic  ne- 
crotic  enteritis  is  present  which  in  rabbits  is  regularly  localized  in  the  appendix  and  ce- 
cum,  while  in  dogs  the  entire  intestinal  tract  and  especially  the  duodenum  is  attacked, 
and  in  monkeys  the  lower  part  of  the  intestine  is  involved. 

The  associated  nervous  manifestations  are,  according  to  experiments  of  Dopter, 
referred  to  changes  in  the  spinal  cord  itself.  These  are  of  a  nature  similar  to  acute 
anterior  poliomyelitis.  Occasionally  a  polio-encephalitis  is  added. 

An  antitoxic  dysentery  serum  is  obtained  by  immunization  of 
Dysentery      horses  and  goats.     Various  methods  have   been   employed 
Serum.        to  obtain  it.     Of   the  older  authors,  Shiga  and  Kruse  im- 
munized animals  with  dysentery  bacteria  and  thus  produced 
a  serum   which  possessed  besides  its  bacteriolytic  and   agglutinating 
properties  also  a  weak  antitoxic  action.     Rosenthal,  Todd,  Kraus  and 
Doerr  employed  the  toxin  itself  for  immunization  purposes. 

In  standardization  of  the  serum  the  properties  to  be  determined  are 
three.  [Kraus  and  Doerr  employ  rabbits  in  this  work.] 

1.  Its  power  of  neutralizing  toxin  in  vitro. — Toxin  and  antitoxin  are 
mixed  in  various  proportions;   the  mixtures  allowed  to  stand  fifteen 
minutes  at  room  temperature  and  then  injected  intravenously. 

2.  Its  power  of  neutralizing  toxin  in  vivo. — The  toxin  is  injected  into 
the  right  vein  and  the  antitoxin  at  the  same  time  into  the  left  vein. 

3.  Its  curative  power. — The  antitoxin  is  injected  at  various  intervals 
after  the  toxin. 

These  three  therapeutic  factors  do  not  appear  simultaneously.  The  power  of 
neutralization  in  vitro  is  first  in  evidence.  Only  very  much  later  does  the  serum  de- 
velop its  curative  strength  and  ability  to  neutralize  in  vivo. 

In  animal  experimentation,  the  antitoxic  serum  exhibits  its  neutralizing  and  cura- 
tive properties  only  if  injected  intravenously. 

Dysentery  serum  has  been  employed  with  fairly  good  results.  Only 
infections  caused  by  the  Shiga-Kruse  bacilli  can,  however,  be  benefited. 
The  serum  should  be  given  subcutaneously  and  as  early  in  the  stage  of  the 
disease  as  possible.  The  dose  advised  by  the  authors  varies  greatly, 
on  account  of  the  difference  in  strength  of  the  numerous  sera  and 
the  severity  of  the  infection.  In  cases  of  moderate  illness,  it  is  as  a 
rule  sufficient  to  give  one  to  two  injections  of  20  c.cm.  of  a  strong  antitoxic 
serum  which  can  neutralize  toxin  both  in  vivo  and  in  vitro.  Vaillard  and 
Dopter  have  injected  as  many  as  80  to  100  c.cm.  in  the  severer  cases. 

The  good  effect  of  the  serum  manifests  itself  by  an  improvement  in 
both  the  general  and  local  symptoms.  If  high  fever  exists,  the  tempera- 
ture sinks.  If  collapse  temperature  is  present,  it  usually  rises.  The 
subjective  complaints,  especially  the  sleeplessness,  improve.  The  blood 


TOXIN  AND  ANTITOXIN 


in  the  stools  disappears;  the  movements  of  the  bowels  become  less  fre- 
quent and  the  severe  pains  are  absent.  Finally,  the  consistency  of  the 
stools  changes  and  at  the  end  becomes  normal. 

Prophylactic  use  of  the  serum  has  met  very  favorable  confirmation  in 
the  work  of  Kruse,  Vaillard  and  Dopter,  and  Rosculet.  Rosculet's 
statistics  are  especially  interesting.  In  1905  during  a  dysentery  epidemic 
in  Roumania,  Rosculet  injected  eighteen  apparently  healthy  individuals 
living  at  the  homes  where  dysentery  cases  existed,  with  5  c.cm.  of  the 
serum.  Eighteen  similar  patients  were  removed  from  the  dysentery 
surroundings,  but  received  no  serum.  The  results  were  that  of  the  first 
group  no  fresh  cases  of  infection  arose,  while  of  the  control  group  fourteen 
became  infected. 

It  is  rather  premature  to  determine  definitely  the  value  of  the  dysentery 
serum  therapy;  enough  has  been  seen,  however,  to  advocate  its  use  when- 
ever possible. 

Staphylolysin,  or  Staphylohemotoxin. — According  to  the  experi- 

Staphyloly-  ments  of  M.  Neisser  and  Wechsberg  the  pyogenes  staphylo- 

sin.        cocci  produce  a  typical  hemolysin  which  is  identical  for  both 

the  aureus  and  albus  cultures.     By  immunization  with  this 

hemotoxin,    an    antihemotoxin    (antilysin)    is    obtained.     Neisser    and 

Wechsberg  further  discovered  that  human  serum  and  serum  of  certain 

animal  species  normally  contained  antistaphylolysin ;  less,  however,  in 

amount  than  immune  sera.     Working  on  the  principle  that  in  staphylo- 

coccus  diseases,  a  hemotoxin  is  formed  which  incites  the  development  of 

antihemotoxin  for  the  protection  of  the  animal,  Bruck,  Michaelis  and 

Schulze  attempted  to  employ  the  presence  of  antistaphylolysin  in  the 

serum  as  evidence  of  the  existence  of  staphylococcus  infections. 

As  Staphylolysin,  a  twelve  to  thirteen  day  old  bouillon  culture  of 
freshly  isolated  staphylococcus  pyogenes  serves  very  well.  This  can  be 
preserved  by  adding  5  c.cm.  of  the  following  mixture  to  100  c.cm.  of  the 
bouillon  filtrate:  10  carbolic,  20  glycerin,  70  aqua.  The  hemotoxin  content 
is  approximated  according  to  the  following  scheme: 


Amount  of  filtrate 

Fresh  rabbit  blood 

Physiological 
NaCl  solution 

Result  of  hemolysis  after  2  hours 
in  incubator  at  37°   C.  and  24 
hours  in  ice  box 

0.2      c.cm. 

i  drop.              up  to  2  c.cm. 

complete. 

o.  i      c.cm. 

i  drop.            '.  up  to  2  c.cm. 

complete. 

0.05    c.cm. 

i  drop. 

up  to  2  c.cm. 

complete 

0.025  c.cm. 

i  drop. 

up  to  2  c.cm. 

complete. 

o.oci  c.cm. 

i  drop. 

up  to  2  c  cm. 

incomplete. 

0.005  c.cm. 

i  drop. 

up  to  2  c.cm. 

layer  of  red  blood  cells. 

STAPHYLOLYSIN   CONTENT   OF    SERUM 


93 


Thus  0.025  is  the  smallest  dose  which  can  completely  hemolyze  the  given 
quantity  of  red  blood  cells. 

The  amount  of  antilysin  is  estimated  by  adding  varying  amounts  of 
serum  to  the  constant  minimal  hemolytic  dose  of  the  staphylolysin  and 
determining  what  amounts  of  serum  contain  enough  antilysin  to  prevent 
hemolytic  action  of  the  staphylolysin.  It  is  best  to  allow  the  staphylolysin 
and  serum  to  remain  mixed  for  some  time  before  adding  the  rabbit  blood, 
so  as  to  give  the  antitoxin  a  chance  to  neutralize  the  toxin. 

As  every  normal  serum  contains  a  certain  amount  of  antilysin,  it  is 
necessary  in  order  to  obtain  the  pathological  variations,  to  use  a  normal 
serum  as  a  control.  Such  a  serum,  o.i  c.cm.  of  which  just  suffices  to  neu- 
tralize twice  the  minimal  hemolytic  dose,  was  dried  in  vacuum  and  used  by 
Bruck,  Michaelis  and  Schulze,  as  standard  serum. 

Estimation  of  antilysin  content  of  the  standard  serum: 


Twice  the  minimum  hemolytic 
toxic  dose. 

0.05 

0.05     • 

0.05 

0.05 

0.05 

Standard  normal  serum  

O.  2 

O.  I 

0.05 

0.025 

O.OI 

Result  after  24  hours 

No 

No 

Slight 

Moderate 

Complete 

hemolysis. 

hemolysis. 

hemolysis. 

hemolysis. 

hemolysis. 

These  mixtures  were  allowed  to  stand  for  one  hour  at  37°  C.  and  then  i 
drop  of  rabbit's  blood  was  added,  allowed  to  remain  for  two  hours  at  37°  C. 
and  twenty-four  hours  in  the  ice  box.  :' 

The  standard  serum  was  always  freshly  prepared  in  the  form  of  a  10 
per  cent,  solution  in  distilled  water  (o.i  :  i). 

In  the  above  manner  the  antilysin  content  of  the  serum  from  patients 
with  distinct  or  suspicious  staphylococcus  infections  was  estimated.  The 
completely  neutralizing  dose  of  the  standard  serum  (o.i  above)  was  taken 
as  i  and  the  neutralizing  dose  of  the  serum  for  examination  compared 
with  this;  if  0.05  c.cm.  of  a  serum  x  neutralized  the  same  amount  of  toxin 
as  o.i  of  standard  serum,  the  antilysin  value  of  the  serum  x  was  2. 

From  the  comparative  studies  of  Bruck,  Michaelis  and  Schulze  it  was 
concluded  that  most  of  the  normal  sera  had  values  ranging  from  i  down; 
occasionally  results  as  high  as  5  were  obtained.  Out  oj  twenty -five  cases  of 
staphylococcus  infections  nineteen  gave  values  varying  from  10  to  100.  Fig- 
ures as  high  as  these  can,  according  to  these  authorities,  become  of  valuable 
aid  in  diagnosis. 

Although  these  findings  were  corroborated  by  Arndt  and  others,  the 
method  cannot  as  yet  be  classed  among  those  of  clinical  diagnostic 
importance.  Similar  study  of  other  infections  has  not  been  undertaken. 


94  TOXIN  AND  ANTITOXIN 

In  addition  to  the  toxins  reviewed  in  these  chapters,  recent  work  has 
proven  that  toxins  may,  under  certain  conditions,  be  derived  from  bacteria 
other  than  those  mentioned,  e.g.,  cholera,  typhoid  bacteria  and  meningo- 
cocci.  Problems  such  as  these  are  still,  however,  open  to  scientific 
discussion;  consequently  no  exact  statements  can  be  made  here. 


CHAPTER  IX. 

THE  TOXINS  OF  THE  HIGHER  PLANTS  AND  ANIMALS  AND  THEIR  ANTI- 
BODIES.    FERMENTS  AND  ANTIFERMENTS. 

The  toxins  thus  far  studied  were  all  secretory  products  of  bacteria. 
This  power  of  forming  toxins  is  not,  however,  limited  to  bacteria  alone, 
as  there  is  a  class  of  higher  plants  and  animals  that  produce  characteristic 
poisons  against  which  immunization  can  be  undertaken  and  an  antitoxic 
serum  obtained.  Aside  from  snake  poison,  the  members  of  this  group 
bear  little  practical  medical  interest.  The  detailed  study  of  these  plant 
toxins  (Phytotoxin)  and  those  of  animal  origin  (Zootoxin)  has,  however, 
greatly  increased  the  theoretical  knowledge  of  the  phenomena  of  reaction 
and  immunity. 


Phytotoxins. 

The  most  important  phy  to  toxins  are: 

1.  Ricin. 

2.  Abrin. 

3.  Crotin. 

Ricin  is  a  deadly  poison,  of  which  the  smallest  fractions  of  a  milligram 

Ricin.       are  sufficient  to  kill  rabbits.    Like  bacterial  toxins,  ricin  requires  for  its 

action  an  incubation  period  of  at  least  twenty-four  hours.     The  typical 

postmortem  findings  consist  of  redness  and  swelling  of  Peyer's  patches.     Ricin  is  a 

hemo toxin;  if  mixed,  as  an  emulsion,  with  red  blood-cells,  the  erythrocytes  sink  to  the 

bottom  and  are  agglutinated. 

Ehrlich  succeeded  in  immunizing  animals  against  ricin  by  first  giving  it  to  them  per 
os  in  increasing  doses  for  a  long  period  of  time,  and  later  on  by  subcutaneous  injection. 
The  antitoxic  serum  thus  produced  neutralizes  the  poisonous  action  of  ricin  both  in 
vivo  and  in  vitro. 

Abrin,  a  vegetable  poison,  is  obtained  from  jaquirity  (Abrus  precatorius) 
Abrin.       and  in  its  action  closely  resembles  ricin,  but  is  less  poisonous.     It  is  a 
marked  irritant  of  the  conjunctiva  and  was  at  one  time  employed  in 
cases  of  trachoma. 

Roemer  found  that  by  repeated  instillation  of  abrin  into  the  same  conjunctival  sac, 
no  reaction  was  ultimately  obtained  (local  immunity),  while  the  conjunctiva  of  the 
other  eye  retained  its  susceptibility.  If  the  instillation  was  continued  for  a  long  period 
of  time,  a  "general  immunity"  was  attained  which  extended  to  the  conjunctivas  of 
both  eyes.  As  a  result,  in  the  serum  of  such  animals  antiabrin  could  be  demonstrated. 

95 


96  THE  TOXINS  OF  THE  HIGHER  PLANTS  AND  ANIMALS 

Crotin  is  the  seed  of  croton  tiglium,  a  substance  less  poisonous  than 

Crotin.      either  ricin  or  abrin.     It  does  not  agglutinate,  but  produces  hemolysis 

of  rabbits'  red  blood  cells.     Toward  the  red  blood  cells  of  other  species 

(e.g.,  bird),  it  is  entirely  inactive.     The  immunization  of  Babbits  is  readily  brought 

about  by  subcutaneous  injections.     Their  serum  neutralizes  the  hemotoxic  action  in 

vitro. 


The  Zootoxins. 

Most  important  of  the  animal  toxins  are 

1.  Phrynolysin   (toad  poison).      ]  _ 

A       T_     i     .     /    .,          .      \      \  Simple  hemo toxins. 

2.  Arachnolysm  (spider  poison),    J 

3.  Snake  poison.  }  _ 

0        .  .  Lecithin  producing 

4.  Scorpion  poison,  >  _  . 

-n  hemo  toxins. 

5.  Bee  poison, 

The  one  striking  characteristic  of  toxins,  that  an  immunity  can  be 
raised  against  them,  is  also  possessed  by  these  poisons.  Aside  from  this  fact 
they  present  many  variations  from  the  true  class  of  toxins.  Most  of  these 
poisons  are  complex,  i.e.,  they  contain  more  than  one  toxin  and  all  are 
hemotoxic. 

Toad  poison  is  obtained  by  rubbing  up  the  skins  of  the  Bombinator  igneus;  spider 
poison  by  trituration  of  the  living  "cross  spiders"  (Epeira  diadema)  in  three  or  four 
times  the  amount  of  physiological  salt  solution  containing  toluol. 

The  toad  and  spider  poisons  contain  simple  hemotoxins,  that  is  to  say,  by  the  mixture 
of  small  amounts  of  this  toxin  with  erythrocytes  absolutely  serum-free,  hemolysis  of 
the  latter  takes  place.  Not  all  species  of  blood  are  affected  alike.  The  red  blood 
corpuscles  of  sheep,  goats,  and  rabbits  are  especially  adapted  for  experiments  with 
phrynolysin,  while  rabbits',  rats',  and  human  blood  is  more  suitable  for  arachnolysin. 
Immunity  of  rabbits  is  easily  attained. 

Snake  Poisons. 

The  most  familiar  poisonous  snakes  are  the  Cobras  (Naja)  of  India  and 
Indo-China  which  belong  to  the  family  of  Colubridae,  the  European  viper, 
and  the  American  rattlesnake;  the  last  two  being  of  the  Viperidae  species. 
The  poisons  of  these  two  families  show  great  individual  differences.  Thus, 
those  of  the  Colubridae  group  are  decidedly  thermo-resistant  (temperatures 
as  high  as  100°  C.)  while  the  viper's  poison  is  entirely  destroyed  at  a  tem- 
perature varying  between  80  to  85°  C.,  and  markedly  weakened  at  7o°C. 

Snake  poisons,  as  a  rule,  produce  both  local  reactions  at  the  point  of  the 
bite,  and  severe  general  disturbances. 

The  cobra  bite  is  only  slightly  painful.  A  characteristic  feeling  of  stiffness  extends 
from  the  point  of  infection  over  the  entire  body.  In  several  hours  a  rapidly  increasing 
weakness  sets  in  terminating  in  deep  coma  and  death. 


COBRA   HEMOLYSIS  97 

The  viper  bite  incites  a  very  severe  local  reaction.  The  point  of  infection  is  red, 
extremely  painful  and  swollen.  Convulsions,  hemorrhages,  followed  by  delirium 
which  finally  changes  into  stupor  are  manifest,  and  death  takes  place  in  one  to  three 
days.  If  the  poison  gets  into  the  circulation  directly,  death  is  likely  to  occur  in  a  few 
minutes. 

The  prognosis  of  a  snake  infection  depends  largely  upon  the  situation  of 
the  bite.  The  greater  the  blood  supply  of  the  infected  area  the  more 
dangerous  is  the  result.  Bites  received  through  the  clothing  are  relatively 
less  dangerous,  as  a  great  part  of  the  poison  remains  adherent  to  the 
clothing. 

Snake  poisons  act  primarily  upon  the  nervous  system  and  blood, 
although  they  exhibit  a  number  of  other  toxic  and  ferment  properties. 
Thus  viper  toxin  occasions  immediate  coagulation  of  the  blood  by  its 
action  upon  the  vascular  endothelium  and  has  for  this  reason  been  called 
by  Flexner  and  Noguchi,  "Hemorrhagin." 

Furthermore,  all  snake  poisons  have  a  hemolytic  power. 

Cobra  hemolysis  represents  one  of  the  most  interesting  of 

Cobra      biological  phenomena,  and  since  it  may  possibly  be  employed 
Hemolysis.  in  clinical  methods  of  examination  its  action  will  be  here 
reviewed. 

Cobra  hemotoxin  is  characterized  by  its  power  of  dissolving  the  red 
blood  corpuscles  of  certain  kinds  of  animals  (ox,  sheep  and  goat)  only  in 
the  presence  of  serum.  Other  red  blood  cells  do  not  require  any  serum 
for  their  hemolysis  (dog,  guinea-pig,  man,  rabbit,  horse).  If  the  red 
blood  corpuscles  of  the  first  group  of  animals  washed  free  of  their  serum 
are  mixed  with  cobra  poison,  no  hemolysis  takes  place.  On  subse- 
quent addition  of  any  fresh  serum,  hemolysis  is  in  evidence.  (Flexner, 
Noguchi.) 

The  agent  which  activates  the  hemolytic  substance  belongs  undoubt- 
edly to  the  class  of  lipoids.  Of  these,  lecithin  stands  pre-eminent.  It  is, 
however,  by  no  means  certain  whether  that  is  the  only  or  the  most  im- 
portant activator. 

Some  sera  exhibit  this  activating  influence  only  when  first  heated.  In 
their  unheated  state  they  are  entirely  inactive.  Other  sera  act  in  a 
manner  decidedly  the  reverse.  Kyes  and  Sachs  mention  that  this  depends 
altogether  upon  the  nature  of  the  lecithin  union.  The  following  table 
shows  the  various  combinations  and  their  resultant  action. 


98 


THE  TOXINS  OF  THE  HIGHER  PLANTS  AND  ANIMALS 


Combination 

Power  of  serum  to  activate  the  hemolysis 

Serum 

Red  cells 

If  serum  is 
unheated 

If  serum  is  heated  at 

56°  C. 

65  to  100°  C. 

Horse 

Ox. 

! 

I 

+weak  hemolysis. 

Horse  
Ox  

Horse  

Horse  

Ox 

Ox. 

Sheep 

Ox 

Sheep  

Sheep  

Human 

Ox. 

Human 

Human 

Rabbit  

Ox  

Guinea-oiff 

Ox.. 

Guinea-pig 

Rabbit 

-{-Signifies  hemolysis. 
—  Signifies  no  hemolysis. 


Cobra  Hemolysin  Test. 

1.  Washing  of  Erythrocytes. — The  blood  is  collected  into  sterile  flasks  containing 
sterile    glass  beads.    It  is  shaken  and  thus  defibrinated  to  prevent  coagulation. 
The  defibrinated  blood  is  next  centrifugalized  and  the  serum  separated  and  drawn 
off  with  a  pipette.    The  red  blood  cell  sediment  is  mixed  with  physiological  salt  solu- 
tion and  again  centrifugalized.    This  procedure  is  repeated  several  times  until  all  the 
serum  is  removed.    The  red  blood  cells  are  used  in  a  5  per  cent,  suspension;  i.e.,  i 
part  of  washed  erythrocytes  suspended  in  19  parts  of  saline. 

2.  The  Activating  Agent. — As  an  activating  agent  0.2  c.cm.  of  serum  or  a  o.i  per 
cent,  lecithin  solution  is  employed.    The  lecithin  can  be  kept  as  a  stock  solution 
consisting  of  i  g.  lecithin  in  100  c.cm.  of  methyl  alcohol.    A  o.i  per  cent,  solution 
of  the  stock  mixture  is  made  by  mixing  o.i  c.cm.  of  the  solution  with  9.9  c.cm. 
of  physiological  salt  solution. 

3.  The  snake  poison  hemotoxin  is  resistant  toward  heat  so  that  it  may  be  heated 
to  almost  70°  C.  without  interfering  with  its  activity.     Cobra  poison  contains  the 
greatest  amount  of  hemotoxin.    While  i  mg.  of  cobra  toxin  hemolyzes  i  c.cm.  of  5 
per  cent,  horse's  red  blood  cells  in  five  to  ten  minutes,  a  similar  amount  of  viper  toxin 
requires  thirty  minutes  for  the  same  action. 

V.  Dungern  and  Coca  explain  this  type  of  hemolysis  by  the  existence 
of  a  ferment  within  the  snake  poison  which  breaks  up  the  lecithin  with 
the  liberation  of  oleic  acid.  This  acid  has  long  been  known  as  a  hemo- 
lytic  agent.  The  necessity  for  adding  lecithin  or  serum  to  certain  species 
of  blood  is  explained  by  the  variability  in  the  lecithin  content  of  the 
erythrocytes,  or  a  variability  in  the  lecithin  union. 


MUCH  AND   HOLZMANN   "  PSYCHO-REACTION  99 

Calmette  notes  in  the  blood  of  tuberculous  patients  more  than  the  nor- 
Cobra  loxm       ,  ri    ...      .       .  .    .  .  .  . 

.     .      .        mal  amount  of  lecithin;  for  that  reason  their  serum  can  be  used  in  very 

small  doses  to  activate  the  cobra  hemolysin.     By  this  means  he  attains 

,    .    a  diagnostic  reaction  for  tuberculosis.     On  examination  of  the  blood  of 
luberculosis.  .         ,      .    ,.  . ,     .    .     . 

177  tubercular  individuals  he  has  found: 

78  per  cent,  of  positive  reactions  in  the  first  stage  of  tuberculosis. 

57  per  cent,  of  positive  reactions  in  the  second  stage  of  tuberculosis. 

70  per  cent,  of  positive  reactions  in  the  third  stage  of  tuberculosis. 
Szaboky  has  confirmed  these  findings,  but  not  enough  control  examinations  of  normal 
individuals  or  of  other  infections  had  been  made  to  firmly  establish  the  diagnostic 
value  of  the  test. 

At  the  instigation  of  the  author,  Alessandrini  repeated  the  work  of 
Calmette  but  came  to  different  conclusions.  The  reaction  was  not 
found  specific  for  tuberculosis;  it  was  given  by  various  other  diseases. 
Furthermore  Alessandrini  could  not  confirm  the  hypothesis  that  hemolysis 
was  dependent  upon  the  lecithin  or  lipoid  content  of  the  serum.  By 
simple  mixture  of  cobra  poison  and  horse's  erythrocytes,  he  found  that 
the  resistance  of  the  cells  toward  any  traumatism  becomes  greatly 
diminished  so  that  hemolysis  can  be  attained  even  by  hypotonic  salt 
solutions  in  concentrations  which  under  normal  circumstances  are  entirely 
ineffective  for  horse's  red  cells. 

The  hemolysis  of  snake  poison  can  be  overcome  or  interfered  with  by  the 
addition  of  large  amounts  of  normal  serum,  cholesterin,  and  small  amounts 
of  snake  poison  serum. 

Much  and  Holzmann  have  recently  described  the  so-called  "Psycho- 

The  Psycho-  reaction"  which  can  be  explained  thus — Normal  serum,  when  added  to 

reaction,     a  mixture  of  cobra  extract  and  human  red  blood-cells  will  not  interfere 

with  consequent  hemolysis.  If,  however,  the  serum  is  obtained  from 
patients  suffering  from  depressive  mania,  circular  insanity  or  dementia  praecox,  and 
added  to  the  cobra  extract  and  human  red  blood-corpuscles,  the  expected  hemolysis 
does  not  take  place.  One  would  naturally  suppose  that  this  fact  would  be  employed 
for  clinical  diagnosis,  but  unfortunately  it  has  been  generally  proven  by  most  authori- 
ties that  it  is  altogether  impossible  to  do  so  for  the  simple  reason  that  it  is  not  abso- 
lutely specific.  Bauer  has  found  the  same  reaction  with  navel  blood.  It  is  probable 
that  the  interference  with  hemolysis  is  brought  about  by  an  increase  in  the  cholesterin 
of  the  serum — a  greater  possibility  in  diseases  of  the  central  nervous  system  than 
in  any  physiological  or  other  pathological  condition. 

Cobra      In  immunizing  laboratory  animals  one  cannot  start  with 
Immunity,  inoculations  of  the  unaltered  snake  poison. 

Phisalix  and  Bertrand  begin  with  subcutaneous  injections  of  a  toxin  heated  to 
75°  C.  and  after  two  days  use  one-half  of  the  minimal  lethal  dose  of  the  unaltered 
toxin. 

Calmette  weakens  the  cobra  poison  by  the  addition  of  an  equal  amount  of  r  per 
cent,  gold  chlorid,  and  after  four  such  injections  with  increasing  amounts  at  each  time, 
the  pure  toxin  in  very  small  doses  is  employed. 


IOO  THE  TOXINS   OF  THE  HIGHER  PLANTS  AND  ANIMALS 

In  the  same  manner  Calmette  immunized  horses  and  obtained  highly 
antitoxic  sera.  He  tested  the  strength  of  these  sera,  as  follows: 

1.  Upon  Rabbits. — Each  animal  received  an  injection  of  2  c.cm.  of  the  serum  into 
the  vein  of  one  ear,  and  after  two  hours  i  mg.  of  toxin  into  the  vein  of  the  other  ear.     A 
control  animal  was  similarly  treated  with  toxin  only.     The  latter  animal  died  in  a  half 
hour,  while  the  former  remained  alive. 

2.  Upon  While  Mice,. — Diminishing  amounts  of  serum  were  mixed  in  test  tubes, 
with  o.oooi  gm.  of  toxin  (in  i  per  cent,  solution)  and  the  mixtures  injected  into  the  mice. 
The  amount  of  serum  which  should  completely  neutralize  the  toxin  must  not  be 
more  than  0.03  c.cm. 

According  to  Calmette  one  can  judge  the  efficiency  of  an  immune 
serum  by  its  antihemolytic  power,  inasmuch  as  the  hemotoxic  and 
neurotoxic  actions  run  parallel.  This  is  denied  by  Noguchi. 

The  scorpion  and  bee  poisons  display  properties  similar  to  those  of  the 
cobra  poison.  They  also  combine  with  lecithin  to  produce  hemolysis. 

Thus  far,  it  has  been  shown  that  the  lipoids,  especially  lecithin  are 
actively  associated  in  the  hemolysis  of  erythrocytes;  whether  the  toxin 
combines  with  the  lipoids  and  forms  a  toxolipoid  (toxolecithid)  which  is 
hemotoxic,  or  whether  as  v.  Dungern  believes,  the  hemolytic  action  is  due 
to  the  fatty  acid  derived  from  the  lecithin  by  the  ferment  action  of  sub- 
stances contained  in  the  poison,  has  not  been  definitely  proven. 

Quite  recently  it  has  been  thought  that  pernicious  anemia  and  parox- 
ysmal hemoglobinuria  are  closely  associated  with  such  toxolipoids. 


Tallquist  obtained  from  a  Bothriocephalus  latus,  a  hemotoxic 

Pernicious  poison  of  a  lipoid  nature  which  experimentally  produced  a 

Anemia,     blood  picture  characteristic  of  pernicious   anemia.     But  it 

would  be  incorrect  to  associate  all  forms  of  pernicious  anemia 

with  tape- worm  poison;  more  probable  is  it  that  hemo toxins  are  formed 

within  the  organism  itself. 

In  paroxysmal  hemoglobinuria  a  hemotoxin  of  very  peculiar 
Paroxysmal    properties  is  found  circulating  in  the  blood. 
Hemoglo-     It  can  be  demonstrated  as  follows. 

binuria.  x>  Ehrlich's  Method. — One  of  the  patient's  fingers  is  tightened 
by  means  of  a  small  tourniquet  and  kept  immersed  in  ice-cold 
water  for  half  an  hour.  Some  blood  is  then  collected  into  a  capillary  pipette, 
from  the  finger  thus  tied,  and  as  a  control,  blood  from  a  finger  of  the  other 
hand  is  drawn  off.  This  is  allowed  to  clot  and  then  centrif ugalized.  The 
results  are  that  the  serum  from  the  finger  held  in  the  ice  water  is  tinged  red 
from  dissolved  hemoglobin  while  the  control  serum  is  normally  pale. 

2.  Donath-Landsteiner' 's  method  repeats  Ehrlich's  experiment  in  vitro. 


SERUM    TEST   FOR   PAROXYSMAL'  ttEMtfGrOBINURlA  IOI 


The  patient's  and  the  control  individual's  'sertfni  are  eUtfe  tftixed  with 
washed  human  erythrocytes  in  various  proportions.  It  does  not  matter 
whether  the  red  blood  cells  are  obtained  from  the  patient  or  normal  individ- 
ual. The  mixtures  are  allowed  to  remain  for  one-half  to  one  hour  in  the 
ice  box  and  then  from  one  to  three  hours  at  a  temperature  of  37°  C.  The 
serum  from  the  paroxysmal  hemoglobinuria  patient  shows  hemolysis. 

A  control  tube  containing  the  same  ingredients,  in  the  same  proportions 
and  maintained  at  either  cold  or  warm  temperatures,  but  not  at  both  in 
succession  as  above,  exhibits  "no  hemolysis. 

The  hemolytic  process  in  this  disease  is  of  a  complex  nature.  In  the 
cold,  one  element  combines  with  the  erythrocytes,  and  at  high  temperature 
another  unfolds  hemolytic  tendencies.  Some  sera  lacking  entirely  or  not 
having  enough  of  the  second  element  in  the  serum,  demonstrate  no  hemoly- 
sis. But  on  addition  of  some  normal  serum  hemolysis  occurs.  It  can  there- 
fore be  concluded  that  the  second  factor  which  acts  in  the  heat  is  present 
within  normal  serum,  while  the  first  substance,  the  specific  one,  is  found 
only  in  the  blood  of  those  suffering  from  paroxysmal  hemoglobinuria; 
(and  according  to  Donath  and  Lands teiner  in  10  per  cent,  of  cases  of  general 
paralysis).  It  is,  in  addition,  the  author's  opinion,  that  similar  toxic 
substances  exist  in  the  blood  of  epileptics  and  idiots. 

Not  all  cases  of  paroxysmal  hemoglobinuria  possess  this  characteristic 
hemotoxin.  In  some  it  is  only  found  periodically. 

No  explanation  has  as  yet  been  offered  for  these  varying  phenomena. 
Attempts  have  been  made  to  ascertain  whether  the  hemotoxin  is  stimu- 
lated by  an  external  agent  or  by  infection  (lues,  malaria,  trypanosomiasis) 
or  whether  it  is  of  endogenous  origin.  The  answer  is  still  for  the  future  to 
disclose. 

The  Antiferments. 

Ferments  are  very  closely  allied  to  toxins  in  their  biological  structure. 
By  the  immunization  of  animals  with  ferments  in  as  pure  a  form  as  possible, 
antiferments  can  be  demonstrated.  Just  like  antitoxins,  antiferments  can 
neutralize  their  respective  ferments  in  vitro.  As  to  their  presence,  it  is 
quite  important  to  know  that  they  are  found  in  normal  serum  in  certain 
small  quantities  (together  with  antitoxins).  The  difference  in  their  pres- 
ence in  a  normal  serum  and  that  in  an  immune,  is  purely  a  quantitative 
one. 

The  antiferments  thus  far  demonstrated  are 

Antilabferment.  Antipepsin. 

Antitrypsin.  Antisteapsin. 

Antifibrinferment. 
//  is  difficult  to  obtain  by  immunization  an  antiferment  serum  of  very  high 


102  THE  TOXINS   OF   THE  HIGHER  PLANTS  AND  ANIMALS 

strength.    Probably  the  normal  organism  is  so  regulated  that  it  compensates 
for  any  increased  amount  of  antiferment. 

Till  recent  times  the  demonstration  of  antiferments  bore  no  clinical 
Antitrypsin.  interest.  The  antibodies  of  the  proteolytic  enzymes  first  began  to  attract 

attention  when  the  inhibitory  influence  which  blood  serum  has  upon 
the  autolysis  of  organs  was  proven.  It  was  Jochmann  and  Muller  who  showed  in 
connection  with  their  studies  of  the  proteolytic  ferments  of  leucocytes,  that  apart 
from  these,  the  serum  itself  possesses  an  inhibitory  influence  upon  the  leucocyte  fer- 
ment. This  is  found  to  be  especially  marked  in  diseases  associated  with  great  destruc- 
tion of  leucocytes.  Following  them,  Marcus,  as  well  as  Brieger  and  Trebing,  discovered 
a  restraining  influence  in  the  serum  upon  the  action  of  pancreas  trypsin  and  proved 
that  the  so-called  antitrypsin  was  considerably  increased  in  carcinoma  patients. 
Bergmann  and  Meyer,  also  working  along  these  lines,  then  demonstrated  that  the 

wrongly  called  "carcinoma  reaction"  was  by  no  means  specific  for  car- 
Brieger's  cinoma,  but  was  found  in  a  large  number  of  other  diseases.  It  cannot, 
Cachexia  as  Brieger  later  announced,  even  be  considered  as  a  criterion  for  cachexia 
Reaction,  (cachexia  reaction). 

Undoubtedly,  the  already  normal  antiproteolytic  power  of  the  serum 
can  be  considerably  increased  in  animal  experimentation  by  a  group  of  well-known  pro- 
teolytic agents,  and  especially  by  leucocyte  ferment  and  pancreatic  trypsin.  To  differ- 
entiate between  antileucocyte  and  antitrypsin  ferment  in  the  narrow  sense  of  the  word, 
is  impossible.  The  one  "immune  serum"  (sit  venia  verbo,  if  one  can  speak  of  immune 
serum  in  this  sense)  neutralizes  the  other  antigen.  Clinically  a  high  and tryptic  titer 
of  the  serum  is  found  in  about  90  per  cent,  of  carcinoma  patients,  and  is  almost  regu- 
larly observed  in  infections  with  high  fevers  as  typhoid,  severe  articular  rheumatism, 
sepsis,  etc.  In  penumonia  there  is  found  during  the  infection  a  marked  change  from 
an  excessively  high  to  a  low  titer.  In  Morbus  Basedowii  (as  well  as  in  experimental 
thyroid  feeding)  it  is  almost  the  rule  to  find  a  high  antitrypsin  content,  but  one  must 
always  keep  in  mind  that  even  few  normal  individuals  show  a  similar  increase. 

The  clinical  diagnostic  importance  of  the  antitrypsin  titer  is  slight  in  comparison 
with  its  experimental  increase.  In  accord  with  the  findings  in  Basedow's  disease,  and  in 
thyroid  feeding  it  may  be  considered  as  an  outcome  of  increased  proteid  destruction 
(hyper-production  of  proteolytic  ferments  in  the  tissues  ?) .  Leucocyte  ferment  has  been 
found  of  practical  use  in  the  treatment  of  cold  abscesses,  i.e.,  in  processes  where  lympho- 
cytosis  and  failure  to  produce  polynuclear  leucocyte  ferment  is  present.  On  the 
other  hand  antitrypsin  or  antileucocyte  ferment  or  even  normal  serum  is  employed  to 
counteract  inflammatory  processes,  i.e.,  to  neutralize  the  excessive  production  of  the 
leucocyte  ferments,  with  apparent  success  (Leucoantifermentin,  on  the  market). 
According  to  recent  findings,  the  antitrypsin  titer  of  the  mother's  blood  increases 
markedly  during  the  period  of  labor,  while  that  of  the  fetus  remains  unaltered. 

There  are  two  methods  for  the  antitrypsin  determination.  The  first 
was  devised  by  Jochmann  and  Muller  for  proving  the  presence  of  leucocyte 
ferment  and  its  antiferment,  and  then  similarly  employed  by  Marcus  in  the 
study  of  pancreatic  ferments.  Its  principle  depends  upon  the  digestive 
action  of  proteolytic  ferments  upon  serum  albumin.  When  a  drop  of 
trypsin  is  placed  upon  a  LofHer's  serum  plate,  after  a  little  while,  a  clear 
spot  appears  where  the  trypsin  was  brought  into  contact  with  the  plate. 


ANTITRYPSIN   DETERMINATION 


I03 


If  to  this  trypsin  an  amount  of  serum  is  previously  added,  which  fully 
neutralizes  the  digestive  action,  no  clear  zone  appears  upon  Loffler's  plate. 

The  details  of  this  procedure  are  as  follows:  The  ferment  solution  consists  of  o.i  gm. 
trypsin,  well  shaken  with  5  c.cm.  of  undiluted  glycerin  and  5  c.cm.  of  distilled  water, 
then  left  in  an  incubator  for  a  half  hour  at  55°  C.,  then  again  shaken  and  filtered. 

The  serum  is  mixed  in  small  test-tubes  or  upon  a  glass  slide  with  varying  amounts 
of  the  trypsin;  thus  i  loopful  of  serum  is  mixed  with  1/2,  i,  2,  3,  4,  etc.,  up  to  20  loopfuls 
of  the  trypsin  solution  and  of  each  of  these  mixtures  one  loopful  is  placed  upon  Loffler's 
plate.  (Ox  serum  plate,  should  be  three  days  old).  The  plates  are  then  placed  into 
the  incubator  for  twenty-one  hours  at  55°  C.  The  presence  or  absence  of  the  clear 
zones  determines  the  quantities  of  ferment  which  respectively  have  not  or  have  been 
neutralized  by  the  one  drop  of  serum  (e.g.,  i :  6  means  that  in  the  mixture  of  6  loopfuls 
of  trypsin  and  i  loopful  of  the  serum  for  examination  the  digestive  power  of  the  trypsin 
was  still  interfered  with). 

The  inequality  in  the  strength  of  the  Loffler  plates,  their  variability  in  the  degree  of 
alkalinity,  the  measurement  by  loopfuls,  all,  might  prove  to  be  sources  of  error  which 
may  greatly  influence  the  results.  Thus  the  latter  can  only  be  taken  as  approximate, 
relative  values. 

The  second,  more  exact  and  satisfactory  method  was  introduced  pri- 
marily by  Gross  and  Fuld  for  presenting  the  action  of  trypsin,  and  was 
modified  by  v.  Bergmann  together  with  Bamberg  and  Meyer  for  the  deter- 
mination of  an ti trypsin.  Numerous  workers  have  found  it  thoroughly 
reliable.  Its  principle  is  based  on  the  digestion  of  a  clear  casein  solution. 
If  the  entire  amount  of  casein  is  digested,  no  more  is  left  to  be  precipitated 
by  the  addition  of  acid  and  therefore  the  solution  remains  clear.  If,  how- 
ever, casein  has  been  left  undigested,  the  addition  of  acid  will  produce  a 
turbid  solution  or  even  a  white  precipitate. 

The  necessary  reagents  are: 

1.  Casein  Solution. — One  gm.  of  casein  is  dissolved  under  slight  heating  in  100  c.cm. 
of  N/io  NaOH;  this  solution  is  next  neutralized  by  N/io  HC1,  litmus  being  used  as 
indicator,  and  diluted  with  physiological  salt  solution  up  to  500  c.cm.     (If  sterilized, 
it  can  be  kept  for  a  long  while.) 

2.  Trypsin  Solution. — 0.5  gm.  of  trypsin  (purissimum  Grubler)  is  dissolved  in  50 
c.cm.  of  normal  NaCL+o.o5  c.cm.  of  normal  sodium  hydrate  solution  and  then  diluted 
with  physiological  saline  up  to  500  c.cm. 

3.,  Acid  Solution. — Five  c.cm.  of  acetic  acid  +45  c.cm.  of  alcohol  +50  c.cm.  of 
water. 

First  the  titration  of  the  trypsin  solution  is  undertaken  in  order  to  find  out  how 
much  trypsin  is  required  to  fully  digest  a  constant  quantity  of  casein.  Gradually 
increasing  amounts  of  trypsin  (from  o.i  to  0.6  c.cm.)  are  placed  in  six  test-tubes  and 
to  each  2.0  c.cm.  of  casein  are  added.  These  tubes  are  placed  in  an  incubator  at 
37°  C.  for  one-half  hour,  and  then  several  drops  of  the  acid  solution  are  placed  into 
each  tube.  The  first  tube,  and  all  those  above  it  that  remain  absolutely  clear, 
contain  enough  trypsin  to  fully  digest  the  2.0  c.cm.  of  casein. 

Now  comes  the  second  part  of  the  test. 

In  each  of  eight  to  ten  test-tubes  are  placed  2  c.cm.  of  the  casein  solution  and  0.5 


104  THE   TOXINS   OF   THE  HIGHER  PLANTS  AND  ANIMALS 

c.cm.  of  a  2  per  cent,  dilution  of  the  serum  for  examination;  to  these  is  next  added  the 
trypsin  solution  in  successively  increasing  amounts,  beginning  with  the  smallest  quan- 
tity which  in  the  first  part  of  the  test  was  sufficient  to  completely  digest  the  given 
amount  of  casein.  Salt  solution  is  then  added  to  each  of  the  test-tubes  so  that  all  con- 
tain an  equal  quantity  of  fluid,  and  the  mixtures  placed  in  an  incubator  at  37°  C.  for 
one-half  hour.  At  the  end  of  this  time,  several  drops  of  the  acid  are  added  to  each  tube. 
Those  tubes  which  become  cloudy  or  show  a  precipitate  designate  the  amounts  of 
trypsin  solution  which  have  been  neutralized  by  the  0.5  c.cm.  of  diluted  serum.  For 
example: 

In  the  first  part  of  the  test  it  was  found  that  the  tube  containing  0.4  c.cm.  of  trypsin 
was  the  first  to  remain  clear,  in  other  words  was  sufficient  to  fully  digest  2  c.cm.  of 
the  casein  solution.  In  the  second  part  of  the  test  the  lower  limit  of  the  added  trypsin 
dilution  was  0.4,  and  it  was  found  that  the  tubes  containing  0.4,  0.5,  0.6,  and  0.7  c.cm. 
of  trypsin,  for  example,  now  gave  precipitates  and  only  0.8  remained  clear.  This 
indicates  that  part  of  the  formerly  sufficient  amount  of  trypsin  was  now  neutralized 
by  the  antitrypsin  of  the  added  serum  so  that  digestion  was  interfered  with.  Thus 
the  antitrypsin  titer  in  this  case  is  0.8. 

Recently  the  above  method  of  trypsin  titration  has  been  applied  to  the  determina- 
tion of  the  presence  of  pancreatic  ferment  in  the  intestinal  secretions,  feces,  and  stomach 
contents. 


CHAPTER  X. 

AGGLUTINATION. 

BACTERIAL  AGGLUTININS.    HEMAGGLUTININS.    TRANSFUSION  TESTS. 

If  the  serum  from  an  immunized  animal,  or  a  patient  convalesc- 
The  Phe-    mg  after  an  infection,  be  mixed  with  a  suspension  of  the  bac- 
o°A ^lu     ter*a  wkicn  were  involved  m  the  production  of  said  conditions, 
tination     a   Peculiar   phenomenon    takes   place.     In    the   former  dif- 
fusely cloudy  liquid,  small  granules  and  clumps  appear  which 
sink  to  the  bottom  of  the  test-tube  and  leave  a  supernatant  clear  fluid.    On 
microscopic  examination,  the  sediment  presents  bacteria,  (which  have  re- 
mained alive  as  is  demonstrable  by  making  cultures  of  same) .     This  same 
observation  can  be  made  with  perhaps  more  flattering  results  when  the 
experiment  is  performed  in  a  hanging  drop.     The  bacteria  are  seen  to  lose 
their  motility,  adhere  to  each  other,  finally  gravitate  toward  larger  groups 
and  arrange  themselves  in  clumps.     The  phenomenon  thus  described  was 
discovered  by  Gruber  and  Durham,  and  is  called  agglutination;  while  sub- 
stances which  cause  this,  agglutinins. 

If  instead  of  the  immune  serum  or  that  of  the  convalescent  patient, 
normal  serum  is  employed  and  the  above  test  repeated,  it  will  be  seen  that 
agglutination  likewise  occurs.  The  reaction  is,  however,  somewhat  incom- 
plete ;  the  clumps  are  smaller,  and  formed  much  more  slowly.  //  a  quantita- 
tive determination  with  different  dilutions  of  both  sera  is  made,  the  power  of 
agglutination  disappears  with  the  normal  serum  at  a  low  dilution,  while  the 
immune  serum  remains  perfectly  active  at  even  much  greater  dilutions. 
Thus  the  main  difference  between  the  agglutinating  normal  and  immune 
serum  is  a  quantitative  one  depending  upon  the  amount  of  agglutinins 
present.  Whether  any  qualitative  difference  exists  between  the  normal 
and  immune  agglutinins  is  doubtful.  It  is,  however,  of  no  practical 
significance. 

If  instead  of  homologous  bacteria,  different  (heterologous)  bacteria  are 
employed,  e.g.,  cholera  vibrio  and  typhoid  serum,  agglutination  also  takes 
place,  if  the  typhoid  serum  is  used  in  concentrated  or  only  slightly  diluted 
form;  but  in  moderate  or  great  dilutions,  no  agglutination  occurs.  Nor- 
mal serum  will  agglutinate  the  cholera  vibrio  in  the  same  strength  as  the 
immune  typhoid  serum.  In  other  words  the  typhoid  serum  contains  more 
agglutinins  for  its  homologous  bacteria  than  a  normal  serum,  but  it  has 
only  the  same  titer  of  agglutination  as  a  normal  serum  for  heterologous 
bacteria. 

105 


106  AGGLUTINATION 

The  agglutination  reaction  is  specific  in  the  respect  that  high  dilutions  of 
serum  will  agglutinate  only  its  homologous  bacteria  and  leave  the  heterologous 
ones  uninfluenced.  Agglutination  becomes  non-specific,  when  concentrated  or 
low  dilutions  of  serum  are  employed. 

The  relative  specificity  just  described  is  of  great  clinical 

Diagnostic  diagnostic  value.     For  example,  given  a  serum  suspicious  of 
Value  of  Ag-  typhoid,  the  question  is  to  establish  this  absolutely.     One 

glutination.  immediately  proceeds  to  make  a  suitable  dilution  of  the 
unknown  serum  and  mixes  with  it  known  typhoid  bacilli. 
A  similar  dilution  of  normal  serum  is  made  as  a  control  and  mixed  with 
the  same  amount  of  typhoid  bacilli.  If  agglutination  occurs  with  the 
unknown  serum  and  not  with  the  control  serum,  the  former  must  have  come 
from  a  typhoid  patient.  If  the  bacteria  are  not  agglutinated,  the  serum 
was  not  of  typhoid  origin. 

In  an  equal  manner  can  the  identity  of  unknown  bacteria  be  estab- 
lished by  the  use  of  known  sera.  Thus,  when  certain  bacteria  have  been 
isolated  and  information  is  wanted  as  to  whether  they  are  typhoid,  an 
emulsion  of  these  is  made  and  mixed  with  a  typhoid  serum  in  suitable 
dilution,  and  a  similar  amount  of  bacteria  is  mixed  with  a  normal  serum  of 
like  dilution.  Agglutination  occurring  in  the  first  of  these  mixtures  and 
not  in  the  second  proves  the  typhoid  character  of  the  unknown  bacteria. 
In  this  manner  the  agglutination  test  can  be  used  for  identification  of  any 
antigen. 

The  practical  application  of  agglutination  has  been  greatly  used 
in  cases  of  typhoid  fever.  Here  agglutinins  are  very  easily  stimu- 
lated in  the  course  of  the  disease  and  generally  they  can  be  demon- 
strated in  the  serum  seven  to  ten  days  after  infection.  The  agglutinins 
remain  not  only  during  the  active  stage  of  the  disease,  but  also  during  the 
convalescing  period.  Widal,  the  Parisian  clinician,  was  the  first  to  adopt 
this  agglutination  reaction  for  the  serum  diagnosis  of  typhoid.  It  is  thus 
commonly  known  as*  the  Widal  reaction. 
Technique  of  The  technique  of  the  reaction  is  as  simple  as  its  principle. 

Aggluti-       This  accounts  for  its  wide  adoption.     It  may  either  be  per- 
nation.      formed  macroscopically  or  microscopically  (orientation  test). 

The  Macroscopic  Agglutination  Reaction. 

For  this  reaction  it  is  necessary  to  have 

1.  The  immune  serum  and  a  normal  control  serum; 

2.  A  homogeneous  bacterial  emulsion. 

The  production  of  a  homogeneous  bacterial  emulsion  offers  slight 
technical  difficulties. 

It  can  be  obtained  in  the  following  ways: 


AGGLUTINATION  REACTION 


107 


a.  Bouillon  Culture. — Many  bacteria,  like  typhoid,  paratyphoid,  dysen- 
tery, coli,  etc.,  grow  very  easily  in  broth.  Such  afresh  (twenty-four hours), 
diffusely  turbid  culture  can  be  employed  readily  for  agglutination  purposes. 
In  place  of  live  bacteria,  dead  may  also  be  used — a  fact  which  has  greatly 
added  to  the  practical  application  of  the  test. 

For  preparing  the  latter,  0.5  per  cent,  of  phenol  or  i  per  cent,  of  formalin  (40  per 
cent.)  are  added  to  the  twenty-four  hour  bouillon  cultures.  The  result  is,  that  a 
sediment  of  bacteria  is  formed  from  which  the  supernatant  fluid  should  be  carefully 
poured  off.  The  bacterial  suspension  is  kept  on  ice  and  thoroughly  shaken  before  use. 

Picker  has  in  this  way  prepared  standard  emulsions  of  dead  typhoid  and  paratyphoid 
bacilli  which  are  sold  by  Merck  under  the  name  of  "Ficker's  Diagnosticum." 

For  Widal's  test,  a  small  quantity  of  the  patient's  blood  is  collected  in  a 
capillary  tube  and  the  end  closed  with  sealing-wax.  The  blood  is  allowed  to  clot, 
and  the  serum  to  separate  off.  The  separation  of  the  latter  can  be  hastened  by 
centrifugalization. 

In  practice,  the  Widal  test  as  performed  with  Ficker's  diagnosticum,  is  arranged  as 
follows: 


Bacillus  suspension 

Dilution  of  serum 

Physiological  salt  solution 

Tube  1050  cm 

o  5  c  cm. 

Tube  2   o  5  c  cm 

o  '  5  c  cm  of  i  °  10 

Tube  3,  o.  5  c.cm. 

0.5  c.cm.  of  i  150 

Tube  4  o  5  c  cm 

o  5  c  cm  of  i  •  100 

One  of  four  results  may  be  obtained, 
i.  2. 


Positive  reaction 

Doubtful  reaction 

Negative  reaction 

Worthless  reaction 

i.  No  agglutination  

No  agglutination  

No  agglutination  

Agglutination. 

2.  Marked  agglutination. 

Marked  agglutination 

Very  slight  agglutina- 

Agglutination. 

tion. 

3.  Marked  agglutination. 

Very  slight  agglutina- 

No agglutination  

Agglutination. 

tion. 

r  •:••'•-•'  -'  :%'.•-••  :.';..'  -;:  '•  .-,;.    -_     - 

4.  Slight  agglutination..  . 

No  agglutination  

No  agglutination  

Agglutination. 

It  is  very  advisable  to  make  control  tests  with  normal  serum.  After  mixture  of 
the  various  ingredients  the  tubes  are  placed  in  the  incubator  at  37°  C.  for  two  hours. 
Then  the  results  are  read  off;  the  first  tube  must  show  absolutely  no  agglutina- 
tion, otherwise  (as  seen  in  Division  No.  4  above)  the  entire  test  is  of  no  signifi- 
cance. The  cause  for  such  spontaneous  or  pseudo-agglutination  occurring  in 
tube  i  may  be  found  either  in  the  bacterial  emulsion  or  NaCl  solution.  The  grade 
of  agglutination  is  estimated  by  the  size  of  the  agglutinated  clumps  and  the  rapidity 
with  which  they  are  formed.  The  mild  grades  of  agglutination  are  frequently  over- 
looked by  the  beginner.  For  typhoid  a  positive  reaction  is  one  where  agglutination 


108  AGGLUTINATION 

takes  place  in  the  dilution  of  i  :ioo;  a  positive  result  in  the  dilution  of  i  :  50  can 
only  be  considered  as  probably  positive. 

As  has  been  said,  broth  cultures  may  be  used  for  the  agglutination  test  if  the 
bacteria  grow  diffusely  and  regularly  within  the  bouillon.  This  is  not  the  case,  how- 
ever, with  all  bacteria,  as  for  example,  the  cholera  vibrio  which  produces  a  thin 
pellicle  upon  the  surface  of  the  broth. 


b.  A  gar  Cultures. — Kolle  and  Pfeiffer  have  advised  instead  of  broth 
the  use  of  agar  cultures.  The  bacteria  are  washed  off,  and  an  even  emul- 
sion made  in  physiological  salt  solution,  or  in  a  dilution  of  the  serum  for 
examination. 


The  details  of  the  procedure  are  as  follows: 

Into  a  row  of  test-tubes  is  placed  i  c.cm.  of  various  dilutions  of  the  serum  for  exami- 
nation, e.g.,  i  :  10,  i :  50,  i :  100,  i :  200,  i :  500.  A  normal  serum  is  similarly 
diluted  as  a  control.  One  other  test-tube  is  to  contain  i  c.cm.  of  saline  only. 

A  full  loop  of  an  eighteen  to  twenty-four  hours  old  agar  culture  is  evenly  and  finely 
rubbed  up  in  each  of  the  above  test-tubes  as  follows: 

The  test-tube  is  held  almost  horizontally  in  the  left  hand  between  the  thumb  and 
index  finger;  a  platinum  loop  between  the  thumb  and  index  finger  of  the  right  hand  is 
filled  with  the  bacteria  from  the  agar  culture,  and  placed  in  the  tube  containing  the 
serum  dilutions.  The  bacteria  are  then  gently  and  thoroughly  rubbed  up  on  the 
moistened  wall  of  the  tube  but  not  within  the  fluid.  By  rolling  the  test-tube  slightly,  a 
part  of  the  rubbed  up  bacteria  is  washed  into  the  fluid  and  the  remaining  bacterial  mass 
is  again  triturated.  This  process  is  repeated  until  all  the  bacteria  are  washed  into  the 
fluid.  Thus,  a  homogeneous  suspension  is  obtained. 

The  author  has  found  this  method  of  Peiffer  and  Kolle  most  accurate. 


It  is  worthy  of  note  in  this  connection,  that  the  controls  show  no  clumps 
or  granules.  (Pseudo-agglutination).  There  are  some  bacteria  which 
can  be  evenly  emulsified  only  with  great  difficulty,  while  others  are  very 
easily  agglutinated  even  by  normal  serum.  In  either  case  the  test  is  not 
conclusive. 

For  the  hanging-drop  method,  blood  is  collected  in  a  Wright  capsule  or 
a  small  test- tube-,  6  to  8  drops  of  blood  suffice.  The  blood  is  allowed  to  clot 
or  the  serum  is  hastened  by  centrifugalization.  Four  loopfuls  of  broth  or 
saline  (or  equal  amounts  as  measured  by  a  Wright  pipette)  are  placed  on 
each  of  two  slides.  To  one  of  these  one  loopful  of  the  serum  (or  one  equal 
part  as  measured  by  the  Wright  pipette)  is  added  and  thoroughly  mixed. 
From  this  mixture  one  loopful  or  equal  measure  is  mixed  with  the  broth  or 
normal  saline  upon  the  second  slide;  thus  making  serum  dilutions  of  i  15 
on  the  first  slide  and  i  :  25  on  the  second  slide.  A  loopful  of  typhoid  cul- 
ture is  placed  on  the  center  of  each  of  two  cover  slips.  To  the  first  is 
added  one  loopful  of  the  serum  dilution  1 125,  and  to  the  second  is  added 
one  loopful  of  the  serum  dilution  i  :  5  thus  making  a  dilution  of  i  :  50  and 


PRODUCTION    OF   AGGLUTINATING   SERA  I  Op 

i  :io  respectively.  Each  cover  slip  is  inverted  over  a  hollow  slide  pro- 
tected by  vaseline,  and  examined  microscopically.  A  control  with  normal 
serum  and  also  one  with  culture  alone  should  be  made. 

For  the  identification  of  bacteria  only  highly  agglutinating 
Production  animal  sera  can  be  employed.     Rabbits,  goats  and  horses, 
of  Aggluti-  are  most  suitable  for  such  experiments.     The  best  results 
nating  Sera.  are  obtained  when  the  animals  are  immunized  intravenously 
by  repeated  injections  with  gradually  increasing  doses  of  dead 
bacteria  (killed  at  60°  C.).     Usually  two  to  three  injections  of  1/4  to  i  agar 
culture  of  bacteria  suspended  in  saline  solution  suffice  to  give  an  agglutinat- 
ing titer  of  i  to  5000.     The  serum  should  be  withdrawn  eight  to  ten  days 
after  the  last  injection.     With  typhoid  bacteria  one  may  attain  a  strong 
agglutinating  serum  in  a  rabbit  by  the  intraperitoneal  injection  of  i  c.cm. 
of  a  twenty-four  hours'  live  broth  culture.     This  is  to  be  repeated  in  7  to  10 
days.     As  a  matter  of  course,  the  titer  of  the  serum  should  be  tested  from 
time  to  time,  because  the  height  of  the  antibody  curve  can  only  reach  a 
certain  point.    When  a  sufficient  strength  is  obtained  the  animal  is  bled.    It 
is  not  possible  to  produce  equally  strong  agglutinating  sera  for  all  bacteria. 
The  agglutinins  belong  to  the  class  of  the  more  resistant  serum  sub- 
stances.    By  the  addition  of  one  drop  of  pure  carbolic  acid  they  can  be 
preserved  on  ice  for  a  long  time.     Heating  variously  affects  the  different 
bacterial  agglutinins.     The  agglutinins  for  pest  and  tubercle  bacilli  are 
destroyed  at  56°  C.  while  other  bacteria  are  not  influenced  by  even  higher 
temperatures.     The  animal  from  which  the  agglutinating  serum  has  been 
obtained  also  influences  to  a  great  degree  the  resistance  toward  heat. 
Thus  the  typhoid  agglutinating  serum  derived  from  the  horse  is  much 
more  resistant  than  that  obtained  from  the  rabbit. 

The  Microscopic  (Orientation)  Agglutination  Test. 

This  method  is  especially  of  use,  when  only  small  amounts  of  culture 
or  serum  are  obtainable.  Also,  if  agglutination  is  employed  for  the  quick 
recognition  of  bacteria,  as  for  example,  when  it  is  desirable  to  know  whether 
a  blue  colony  on  a  Conradi-Drigalski-agar  plate  is  typhoid  or  not. 

In  such  a  case  a  drop  of  the  immune  serum  in  the  dilution  of  i  :  50  or 
i  :  100  is  placed  upon  a  cover-glass  held  with  a  Cornet's  forceps,  and  a 
small  part  of  the  bacterial  colony  for  identification  carefully  mixed  with 
this  serum.  As  controls,  a  mixture  is  made  with  salt  solution  and  with 
normal  serum.  If  agglutination  occurs,  small  granules  or  clumps  can  read- 
ily be  seen  with  the  naked  eye  by  holding  up  the  cover-glass  against  the 
light.  The  control  glasses  on  the  other  hand  should  show  only  a  homo- 
geneous turbidity.  These  changes  are  still  more  evident  if  the  mixture  is 
examined  microscopically  in  the  form  of  a  hanging  drop.  (Described, 
p.  108.) 


110  AGGLUTINATION 

Group  Agglutination. 

On  testing  the  titer  of  a  strongly  agglutinating  typhoid  serum,  and  a 
strongly  agglutinating  cholera  serum,  against  typhoid,  paratyphoid,  colon 
and  cholera  bacteria,  the  results  will  be  the  following : 


Agglutination  titer 

Of  typhoid  serum 

Of  cholera  serum 

Against  typhoid  .   .  . 

i  :  2000 

i  :  10 

Against  paratyphoid 

i  :  zoo 

i  :  10 

Against  bacter.  coli  
Against  cholera  .          .         .... 

i  125 
i  :  10 

i  :  10 

i  :  3000 

The  cholera  serum  acts  strictly  in  accordance  with  the  rules  stated 
above  for  specific  agglutinins,  i.e.,  marked  agglutination  with  homologous 
bacteria;  very  weak,  with  heterologous.  The  typhoid  serum  on  the  other 
hand,  although  it  fulfills  the  same  requirements  in  the  main,  nevertheless 
it  manifests  some  important  differences  when  mixed  with  heterologous 
bacteria.  It  has  practically  no  influence  upon  the  cholera  vibrio  with  which 
the  typhoid  bacillus  is  not  at  all  related;  agglutination  of  i  :io  can  be 
attained  even  by  a  normal  serum.  The  colon  bacillus  which  closely  re- 
sembles the  typhoid,  morphologically,  but  which  has  very  different  bio- 
chemical properties,  is  more  strongly  agglutinated,  i  125;  while  the  para- 
typhoid bacillus,  very  much  like  the  typhoid  bacillus  both  morphologically 
and  biologically,  is  agglutinated  even  in  larger  dilations,  i :  100.  This  entire 
phenomenon  is  an  expression  of  the  biological  relationship  of  the  various 
bacterial  groups  and  is  known  as  group  reactions. 

An  understanding  of  group  reactions  is  to  be  found  in  a  more 
Partial  Ag-  complete  conception  of  specificity.  From  this  source  we  have 
glutination.  learned  that  the  difference  in  antibodies  is  influenced  by  the 
dissimilarity  of  the  injected  antigen.  For  example,  the  differ- 
ence between  the  cholera  and  typhoid  agglutination  is  caused  by  the  differ- 
ence existing  in  the  protoplasmic  structure  of  the  respective  bacteria.  As 
these  bacteria,  however,  are  not  constituted  of  a  distinct  chemically  de- 
fined substance,  but  made  up  of  a  mixture  of  various  substances,  there 
may  be  a  number  among  them  which  can  act  as  antigens.  If,  figuratively 
speaking,  there  are  five  different  elements  in  the  body  of  the  typhoid  bacillus 
which  can  act  as  agglutinogens,  i.e.,  antigens,  these  should  be  able  to  form 
according  to  the  law  of  specificity  five  different  agglutinins.  On  mixing  a 
typhoid  serum  with  typhoid  bacilli,  one  brings  together  five  distinct  antigen 
antibody  combinations  and  consequently  complete  and  thorough  action 
results  of  this  union.  A  biological  relationship  of  bacteria  implies  the  exist- 


CASTELLANIS   TEST   FOR   MIXED   INFECTIONS  III 

ence  of  some  common  protoplasmic  constituents.  Expressed  in  the  same 
figurative  manner  the  colon  bacillus  can  be  said  to  have  antigen  number  i 
in  common  with  the  typhoid  and  paratyphoid  bacillus  and  the  paratyphoid 
may  have  antigen  numbers  i  and  2  in  common  with  the  typhoid  bacillus. 
As  a  result,  the  typhoid  serum  will  react  with  colon  bacilli  by  virtue  of  their 
common  agglutinin  number  i,  and  with  paratyphoid  bacilli  through  its 
agglutinins  numbers  i  and  2.  The  other  "partial  agglutinins"  remain 
inactive  on  account  of  the  missing  suitable  agglutinogens. 

The  existence  of  such  partial  antigens  and  partial  antibodies  is  for  some 
bacteria  more  than  of  mere  theoretical  importance.  It  is  even  possible  that 
a  strong  colon  serum  will  agglutinate  no  colon  bacilli  other  than  that  par- 
ticular strain  employed  for  the  production  of  the  serum.  Such  being  the 
case  with  a  number  of  micro-organisms,  the  sera  made  at  present  both  for 
diagnostic  and  therapeutic  purposes  are  polyvalent  (multipartial) .  By 
polyvalent  serum  is  meant  one  which  is  produced  either  by  immunizing 
animals  with  many  different  strains  of  the  same  bacterium,  or  a  mixture  of 
sera  obtained  from  different  animals  immunized  with  various  strains. 

The  practical  importance  of  partial  agglutinins  is  recognized 
Castellani's    in  the  diagnosis  of  mixed  infections.     Castellani  found  that  by 

Test.        the  mixture  of  an  immune  serum  with  its  corresponding  bac- 
teria, the  agglutinins  for  these  as  well  as  the  partial  agglu- 
tinins for  the  heterologous  bacteria  are  absorbed.     On  the  other  hand,  if  the 
same  serum  be  mixed  with  the  heterologous  bacteria,  the  agglutinins  for 
the  homologous  group  are  quantitatively  retained. 

A  practical  example  will  make  this  clearer. 

The  serum  of  a  patient  agglutinates  typhoid  as  well  as  paratyphoid  bacilli,  in  a 
dilution  of  i :  100.  This  may  indicate  one  of  three  possibilities: 

a.  Patient  is  infected  with  typhoid,  but  has  formed  an  exceptionally  large  number 
of  partial-agglutinins  for  paratyphoid  bacilli. 

b.  Patient  is  infected  with  paratyphoid  bacilli,  but  has  formed  at  the  same  time 
many  partial-agglutinins  for  typhoid. 

c.  Patient  has  a  mixed  infection  of  typhoid  and  paratyphoid  and  therefore  formed 
agglutinins  for  both. 

A  decision  in  regard  to  the  above  may  be  reached  according  to  the  following  method 
given  by  Castellani: 

Four  rows  of  test-tubes  are  arranged,  each  row  containing  three  tubes  with  i  c.cm. 
of  serum  dilutions  i :  10,  i :  50,  i :  100  respectively.  In  each  of  the  first  and  second  rows, 
i  loopful  of  typhoid  bacteria  is  emulsified. 

In  each  of  the  third  and  fourth  rows,  i  loopful  of  paratyphoid  B.  bacilli  is  emulsified. 

The  tubes  are  placed  in  the  incubator  for  two  hours,  absence  or  presence  of  agglu- 
tination in  each  test-tube  noted,  and  after  centrifugalization  (which  may  become 
unnecessary  if  the  bacteria  are  strongly  clumped  or  grouped  at  the  bottom  of  the 
tube),  the  supernatant  liquid  is  transferred  to  other  test-tubes  and  kept  in  the  same 
order. 

Then  each  of  the  first  row  receives  i  loopful  of  typhoid  bacilli, 

each  of  the  second  row  receives  i  loopful  of  paratyphoid  B.  bacilli, 


112  AGGLUTINATION 

each  of  the  third  row  receives  i  loopful  of  typhoid  bacilli, 
each  of  the  fourth  row  receives  i  loopful  of  paratyphoid  B.  bacilli, 
All  are  once  more  placed  in  the  incubator  for  two  hours. 

a.  If  typhoid  exists  the  agglutination  titer  in  the  second  part  of  the  test  will  become 
weaker  for  the  typhoid  bacilli  in  the  first  row,  and  weaker  for  the  paratyphoid  B.  bacilli 
in  the  second  and  fourth  rows.     The  titer  in  the  third  row  remains  the  same. 

b.  If  paratyphoid  exists,  the  agglutination  titer  for  typhoid  in  the  first  and  third 
row  becomes  less,  that  for  paratyphoid  in  the  fourth  row  diminishes,  while  the  titer  in 
the  second  row  for  paratyphoid  remains  the  same. 

c.  If  a  mixed  infection  exists,  the  agglutination  titer  in  the  first  and  fourth  row 
diminishes  and  in  the  second  and  third  row  remains  the  same. 

In  this  connection  a  few  exceptions  may  be  mentioned: 

A  serum  which  is  kept  for  a  long  time  frequently  loses  part 
Agglutinoids.  or  even  all  of  its  agglutinating  titer.  Whereas  it  formerly 
agglutinated  in  the  strength  of  1:1000,  it  may  now  become 
inactive  in  dilutions  even  of  i :  10.  The  first  thought  that  arises  in  explana- 
tion of  this  is  that  the  serum  has  perhaps  degenerated  and  the  agglutinins 
were  destroyed.  If,  however,  further  dilutions  are  made,  i :  100  may  show 
mild,  while  i :  500  strong  agglutination.  This,  first  of  all,  demonstrates 
that  agglutinins  are  still  present,  although  diminished  in  amount,  and  sec- 
ond, that  another  substance  has  arisen  which  in  the  stronger  concentrations 
interferes  with  agglutination.  A  simple  experiment  explains  this. 

If  the  test-tube  containing  the  serum  dilution  i :  10  and  the  non-agglutinated 
bacteria  be  centrifugalized,  the  serum  removed  and  the  bacteria  mixed  with  a  known 
strongly  agglutinating  serum,  it  will  be  found  that  the  bacteria  have  become  inagglu- 
tinable.  Substances  of  certain  kinds  have  combined  with  the  bacteria  and  prevented 
them  from  undergoing  agglutination.  These  substances  are  strongly  specific,  acting 
only  upon  homologous  bacteria.  Their  origin  can  also  be  demonstrated. 

An  agglutinating  serum  which  is  heated  to  65°  or  70°  C.  loses  its  agglutinating  power 
but  the  substance  interfering  with  the  subsequent  agglutination  has  remained.  Ehrlich 
explains  the  situation  as  follows:  He  claims  that  agglutinins  are  built  complexly;  that 
they  possess  a  binding  (haptophore)  group  by  means  of  which  they  unite  with  the  bac- 
teria (agglutinogen)  and  a  second  group  (ergophore  or  agglutinophore)  by  virtue  of 
which  agglutination  results.  If  serum  is  kept  for  a  long  period  of  time,  or  exposed  to 
high  temperature,  many  of  the  ergophore  groups  are  rendered  inactive,  while  the 
haptophore  groups  being  more  resistant  remain  and  unite  with  bacteria.  Agglutinins 
possessing  only  their  haptophore  groups  are  known  as  agglutinoids.  They  combine 
with  the  bacteria,  and  still  do  not  agglutinate  them,  but  at  the  same  time  prevent 
other  agglutinins  from  acting.  If  this  old  agglutinoid  and  agglutinin  containing 
serum  is  diluted,  so  few  of  both  of  these  substances  remain  that  the  bacteria  can 
absorb  both,  allowing  the  relatively  few  agglutinins  to  manifest  their  activity. 

It  is  important  to  note  in  this  respect  that  occasionally  even  a  fresh,  highly  valent 
serum  will  present  a  tendency  toward  interfering  with  the  agglutination  processes. 
This  is  also  explained  by  the  existence  of  agglutinoids — a  fact  as  yet  not  definitely 
proven. 

Another  finding,  only  encountered  in  exceptional  cases,  is  me  existence 
of  the  so-called  non-agglutinable  strains  of  bacteria.  These  give  all  the 


AGGLUTININS   IN   TYPHOID   FEVER  113 

characteristics  of  the  general  class  of  bacteria  to  which  they  belong,  but  are 
not  agglutinated  by  their  respective  serum;  as,  for  example,  a  strain  of 
typhoid  bacilli,  which  are  not  agglutinated  by  any  typhoid  serum.  The 
only  positive  proof  that  they  are  typhoid  bacilli  is  the  ability  to  produce  by 
their  injection  into  animals  an  active  immunity  against  fully  virulent 
typhoid  bacteria. 

Non-agglutinable  strains  of  bacteria  can  be  isolated  especially  from  the  lower  ani- 
mals. At  times,  however,  they  regain  their  agglutination  property  when  they  are 
grown  in  artificial  media  and  frequently  subplanted.  Possibly,  the  reason  that  the 
bacteria  become  inagglutinable  at  all,  is  that  they  undergo  immunization  within  the 
organism  against  the  existing  agglutinins.  By  growing  bacteria  in  agglutinating  serum 
for  a  certain  time,  one  can  obtain  non-agglutinable  strains. 

i.  Agglutinins  for  typhoid  and  paratyphoid  A  and  B,  can,  not 
Agglutina-  infrequently,  be  demonstrated  in  the  patient's  serum  as  early 
T^h  kl  as  t^ie  ^i1^  ^ay>  but  as  a  rule,  at  about  the  beginning  of  the 
and  Para-  second  week  of  the  disease.  Moreover,  they  remain  within 
typhoid,  the  serum  for  several  weeks  after  the  illness  and  disappear 
only  gradually.  A  positive  agglutination  test  does  not,  how- 
ever, mean  the  existence  of  the  corresponding  disease.  A  healthy  bacillus 
carrier  can  also  have  an  agglutinating  serum.  Some  cases  of  icterus  catar- 
rhalis  even  give  a  positive  Widal  test.  But  in  order  to  assign  to  this  last 
a  correct  explanation,  one  must  remember  that  typhoid  bacilli  may  remain 
in  the  gall-bladder  for  years  and  thus  lead  to  catarrhal  inflammation  and 
stone  formation. 

Partial  agglutinins  from  coli  infections  must  always  be  considered. 
Some  authorities  mention  a  positive  Widal,  in  connection  with  endocarditis 
maligna,  sepsis,  malaria,  phthisis,  and  miliary  tuberculosis. 

An  absence  of  the  agglutination  test,  especially  at  the  early  part  of  an 
illness,  should  not  influence  a  negative  diagnosis  of  typhoid  too  greatly, 
inasmuch  as  many  cases  are  known  where  the  reaction  appeared  for  the 
first  time  during  the  period  of  convalescence.  In  the  employment  of  this 
test  as  an  aid  for  the  differential  diagnosis  between  several  bacterial  in- 
fections, it  is  best  to  titrate  the  serum  to  its  limit,  as  the  higher  titer  for 
one  class  of  bacteria  generally  speaks  in  favor  of  the  infection  by  the  same. 
Paratyphoid  serum  agglutinates  typhoid  bacilli  only  slightly,  while  true 
typhoid  agglutinates  both  typhoid  and  paratyphoid  bacteria  with  almost 
equal  strength.  In  severe  and  difficult  cases,  Castellani's  test  should  be 
performed.  Paratyphoid  B .  serum  always  gives  the  limit  of  its  agglutinat- 
ing titer  both  with  the  pathogenic  mouse  typhoid  and  hog  cholera  bacillus. 

2.  Cholera. — Only  rarely  has  the  agglutination  test  been  employed  with 
the  serum  of  patients  infected  with  cholera.  On  the  other  hand,  if  cultures 
from  a  cholera  stool  are  made  upon  a  plate,  the  identification  of  the  suspi- 
cious colonies  grown  here  is  regularly  conducted  by  means  of  this  test.  For 

8 


1 14  AGGLUTINATION 

this  purpose,  it  is  very  specific,  as  group  reactions  almost  never  take  place. 
Strong  agglutinating  sera  are  easily  obtained  by  immunization  of  animals. 

3.  Epidemic,  Cerebrospinal  Meningitis. — Agglutination  in  this  disease 
serves  mainly  for  the  identification  of  suspicious  meningococcus  cultures. 
As  has  been  shown  by  Wassermann  and  Kutscher,  some  strains  are  agglu- 
tinated only  after  a  long  period  (twenty-four  hours)  and  at  higher  tempera- 
tures as  56°  C.1 

4.  Dysentery. — The  agglutination  property  is  employed  both  for  testing 
the  serum,  and  identifying  cultures.     The  Flexner  type  of  bacillus  pro- 
duces   agglutinins    more    readily    than    the    Shiga-Kruse.     It    is    also- 
agglutinated  more  readily.     Only  positive  reactions  in  dilutions  of  i :  30 
are  of  diagnostic  consideration.    Occasionally,  partial  agglutination  takes 
place  with  heterologous  dysentery  strains,  typhoid  and  colon  bacteria. 

5.  Pest. — The  reaction  is  very  specific,  but  of  slight  significance,  as  it  appears  only 
on  the  ninth  day;  occurring  with  a  serum  dilution  of  1:3,  it  is  considered  of  positive 
diagnostic  value. 

6.  Malta  Fever. — In  most  instances  the  serum  gives  the  agglutination  reaction  with 
the  micrococcus  melitensis.    Normal  serum  may  give  the  reaction  in  dilution  i :  30, 
so  that  higher  dilutions  only  are  of  aid  in  diagnosis. 

7.  Staphylo-,  Strepto-  and  Pneumococci. — Clinically,  the  agglutination  test  is  never 
employed  in  these  cases. 

8.  Tuberculosis. — Here  the  agglutination  test  is  associated  with  the  difficulty  of 
obtaining  a  homogeneous  tubercle  bacillus  suspension.    This,  however,  is  overcome 
in  one  of  two  ways. 

a.  Arloing-Courmont's  Method  (1898). — The  tubercle  bacilli  are  obtained  in  the  so- 
called  "homogeneous  culture"  form.    S.  Arloing  first  grows  the  bacteria  on  potatoes 
for  a  long  time,  and  then  transplants  them  in  glycerin  bouillon  which  is  shaken 
daily  for  five  minutes.    After  a  number  of  subcultivations,  a  culture  is  obtained  after 
several  months.    This  strain  grows  rapidly  in  a  few  days  and  diffusely  clouds  the  broth. 

Such  a  culture  diluted  with  physiological  saline  solution  is  used  for  the  test.  Here 
small  test-tubes  are  preferable  and  the  ingredients  should  be  mixed  in  the  following 
proportions: 

2  drops  of  serum  -f  10  drops  of  culture  (i :  5) 
i  drop  of  serum  -f-io  drops  of  culture  (i:  10) 
i  drop  of  serum  +15  drops  of  culture  (i:  15),  etc. 

The  tubes  are  well  shaken  and  placed  in  the  incubator.  According  to  Arloing 
and  Courmont,  a  positive  reaction  even  in  the  dilution  of  i :  5  speaks  for  tuberculosis. 
Best  results  are  by  this  means  obtained  in  incipient  and  mild  tubercular  cases;  those 
which  are  farther  advanced  do  not  react. 

b.  Method  of  Koch. — Koch  niters  the  ordinary  tubercle  bacillus  bouillon  cultures, 
dries  the  remnants  upon  the  filter,  and  rubs  them  up  in  an  agate  mortar  with  N/5o 
NaOH  to  a  dilution  of  1:100.    The  solution  is  centrifugalized  and  enough  weak 
HC1  is  added  until  the  reaction  is  only  slightly  alkaline.    The  dilution  is  then  brought 

1  Frequently,  during  even  the  first  days  of  the  disease,  the  patient's  serum  in  a  dilution  of 
i- 10  gives  the  agglutination  test.  This  is  rare  with  higher  dilutions  of  the  serum  as  1-50.  It 
usually  takes  some  time  before  the  agglutination  becomes  evident.  \ 


HEMAGGLUTININS  115 

up  to  1:3000  by  the  addition  of  0.5  per  cent,  phenol  in  normal  saline,  and  kept  for 
twenty-four  hours  in  the  incubator. 

A  somewhat  simpler  procedure  is  to  dilute  new  tuberculin  B.  E.  to  i:  100  with  0.5 
per  cent,  of  carbolic  saline  solution,  centrifugalize  this  for  six  minutes  and  then  dilute 
to  i :  1000.  The  solution  thus  obtained  can  be  preserved  in  the  ice-box  for  fourteen 
days.  Just  before  using,  a  still  further  dilution  of  i :  10  is  made. 

The  agglutination  test  has  not  been  generally  adopted  as  a  method  of  diagnosis. 
The  technique  is  rather  difficult,  and  the  results  not  absolutely  reliable.  The  reason 
for  this  is  that  high  agglutination  values  are  rarely  met  with,  and  slight  ones 
are  found  even  in  normal  individuals.  Then,  too,  the  methods  of  tuberculin  diagnosis 
are  so  much  simpler  that  they  have  been  given  the  preference. 

Koch  himself  advised  the  agglutination  test,  not  as  a  means  of  diagnosis,  but 
rather  as  an  aid  in  tuberculin  therapy.  He  found  that  during  the  treatment  of  tuber- 
culosis with  new  tuberculin  the  agglutinative  power  of  the  patient's  serum  increased. 
He  therefore  took  this  as  an  index  of  the  acquired  immunity.  Further  study,  however, 
convinced  him  that  the  agglutination  cannot  thus  be  interpreted,  so  that  at  the  present 
day  tuberculosis  agglutination  has  no  practical  application. 

10.  Glanders. — Highly  valent  sera  can  be  obtained,  according  to  Kleine,  by  intra- 
venous immunization  of  donkeys  and  goats.  The  serum  serves  for  identification  of  the 
glanders  bacilli.  Kleine  prepares  a  standard  bacterial  emulsion  in  the  following  manner : 
Four  well  grown  glanders  cultures  are  killed  at  60°  C.  and  the  mass  of  bacteria  tritu- 
rated in  2  c.cm.  of  1/2  per  cent,  carbolic-saline  solution.  This  is  then  diluted  in  a 
measuring  glass  so  that  40  to  50  c.cm.  of  carbolic-saline  solution  are  added  for  each 
culture.  The  entire  mixture  is  filtered  through  paper  and  3  c.cm.  are  used  in  each  test. 
Normally,  horses  may  have  an  agglutination  titer  up  to  1 1400.  Glanders  in- 
fected animals  react  as  high  as  i :  2000.  Injections  of  mallein  increase  the  agglutina- 
tion titer.  Experiences  in  this  respect  with  the  human  being  are  still  scanty. 

Hemagglu-  Just  as  injections  of  bacteria  produce  bacterial  agglutinins, 
tinins,      injections  of  erythrocytes  stimulate  the  formation  of  hem- 
Immune  and  agglutinins  which  cause  the  red  blood  cells  to  congregate  in 
Normal.      clumps. 

At  times  the  presence  of  hemagglutinins  is  masked  by  the 
simultaneous  existence  of  hemolysins  which  dissolve  the  red  blood  corpus- 
cles. If,  however,  the  immune  serum  is  heated  to  56*°  C.  the  complement 
is  destroyed,  thus  interfering  with  the  action  of  the  hemolysin  and 
allowing  the  agglutinins  to  exhibit  their  action.  In  other  instances  as 
during  the  immunization  of  rabbits  with  dog's  erythrocytes,  hemagglu- 
tinins are  formed  in  such  great  quantities  that  by  mixing  the  immune 
rabbit's  serum  with  the  dog's  erythrocytes  so  strong  an  agglutination 
occurs  that  the  hemolysins  can  no  longer  attack  the  clumped  erythrocytes. 
The  hemolysis  can  be  demonstrated  only  if  clumping  is  prevented 
mechanically,  by  thorough  shaking  of  the  mixture. 

Also  under  normal  circumstances,  i.  e.,  without  immunization,  the  serum 
of  one  animal  species  can  to  a  certain  degree  agglutinate  (and  hemolyze) 
the  red  blood  cells  of  another  species.  The  quantity  of  these  normal 
hemagglutinins  is  comparatively  small. 


1 1 6  AGGLUTINATION 

Still  more  interesting  is  the  observation  that  there  are  hem- 
Isohemag-  agglutinins  against  the  red  cells  of  different  animals  even  of 
glutinins.  the  same  species,  so-called  Isohemagglutinins  or  Isoagglutinins. 
These  have  thus  far  been  demonstrated  in  the  bloods  of  dogs 
(Von  Dungern)  steers  and  rabbits  (Ottenberg  and  Friedman).  Isoagglut- 
inins in  the  human  serum  were  discovered  independently  by  Landsteiner 
and  Shattock  in  1900.  At  first  the  occurrence  of  isoagglutination  was 
regarded  of  pathological  significance,  but  soon  it  was  shown  that  the 
phenomenon  occurred  with  a  large  percentage  of  normal  bloods.  In  fact 
all  human  bloods  can  be  divided  into  four  sharply  defined  groups  according 
to  the  way  in  which  they  interagglutinate.  The  groupings  can  be  explained 
by  assuming  the  existence  of  two  agglutinins  of  which  the  first  group 
possessed  both,  the  second  one,  the  third  one,  and  the  fourth  neither. 
In  each  case  the  cells  are  susceptible  only  to  that  agglutinin  which  does  not 
exist  in  the  individual's  own  serum.  Thus: 

The  serum  of  the  first  group,  designated  as  group  I,  possesses  the  power 
of  agglutinating  the  red  cells  of  members  of  all  the  other  groups  but  the 
red  cells  of  members  of  group  I  are  not  agglutinated  by  any  human  serum. 
This  group  includes  about  50  per  cent,  of  all  persons  examined. 

The  serum  of  the  members  of  the  second  group  agglutinates  the  red  cells 
of  members  of  the  third  and  fourth  groups.  The  cells  of  members  of  the 
second  group  are  agglutinated  by  sera  of  individuals  of  groups  I  and  III. 
The  serum  of  group  III  agglutinates  cells  of  persons  belonging  to  members 
of  the  second  and  fourth  groups;  its  cells  are  agglutinated  by  sera  of  the 
first  and  second  groups. 

The  fourth  group,  whose  members  are  relatively  rare,  is  characterized 
by  possessing  no  agglutinin  for  human  red  cells  and  by  its  cells  being 
agglutinable  by  the  sera  of  all  other  groups. 

The  group  characteristics  are  permanent  for  each  individual  through- 
out his  life.  When  concentrated,  the  agglutinins  act  almost  instantane- 
ously; when  diluted  they  act  more  slowly.  Agglutination  occurs  in  the 
cold  as  well  as  at  high  temperatures.  The  peculiar  groupings  are  not  only 
permanent  with  the  individual  but  they  are  hereditary.  Von  Dun- 
gern and  Hirschfeld  have  conclusively  proved  that  agglutinins  are 
hereditary  and  follow  the  Mendelian  law.  This  observation  had, 
however,  been  made  long  before  this  in  a  paper  by  Epstein  and  Otten- 
berg (1908). 

With  the  recently  increasing  popularity  of  blood  transfusions,  the 
phenomena  of  isoagglutination  and  hemolysis,  the  two  being  very  closely 
related,  have  attained  a  more  practical  significance.  In  selecting  donors 
for  a  transfusion,  agglutination  and  hemolysis  tests  should  always,  when 
time  permits,  be  made  before  operation.  These  tests  in  vitro  are  usually 
a  safe  guide  as  to  conditions  in  vivo.  That  donor  should  be  chosen  who 


TRANSFUSION  BLOOD   TESTS  1 17 

belongs  to  the  same  group  as  the  patient,  that  is  where  no  interagglutina- 
tion  or  hemolysis  exists.  This  is  advisable  not  only  so  as  to  get  the  best 
results  from  the^  transfusion,  but  also  in  order  to  avoid  any  untoward 
symptoms  or  intoxications  that  are  associated  with  the  intravascular 
agglutination  or  hemolysis  (rise  of  temperature,  dyspnea,  edema,  hemo- 
globinuria) .  The  more  important  of  these  two  factors  is  not  as  yet  clear. 
If  for  a  given  transfusion  a  donor  belonging  to  the  same  class  cannot  be 
obtained,  it  is  safer  to  use  a  person  whose  serum  is  agglutinative  toward 
the  patient's  cells  than  one  whose  cells  are  agglutinated  by  the  patient's 
serum. 

The  materials  necessary  for  the  agglutination  and  hemolysis 
Transfusion  tests  are  as  follows: 

Tests.       (i)  Sterile  syringes  or  needles  for  puncturing  the  vein;  (2) 
i  per  cent,  sodium  citrate  solution  in  0.85  per  cent,  salt  solu- 
tion; (3)  0.85  per  cent,  salt  solution;  (4)  a  test-tube  rack  having 
two  narrow   test-tubes    (4X1/2   in.)  for  each  donor  (numbered);    one 
partly  filled  with  the  sodium  citrate  solution,  one  empty. 

From  each  donor  i  to  2  c.cm.  of  blood  are  aspirated  from  a  vein 
of  the  elbow;  several  drops  of  this  blood  are  allowed  to  flow  into  his  tube 
with  the  sodium  citrate  solution,  the  rest  is  collected  in  his  dry  test-tube. 
This  is  also  done  to  the  recipient,  but  from  him  3  to  4  c.cm.  of  blood 
are  necessary  so  as  to  have  sufficient  serum  for  a  number  of  donors  (10-15). 

The  sodium  citrate  tubes  are  centrifugalized  and  a  sediment  of  the  red 
blood  cells  obtained;  the  supernatant  fluid  is  pipetted  off  and  the  red  cells 
made  up  approximately  to  a  10  per  cent,  suspension  with  the  normal  salt 
solution. 

The  serum  tube  is  also  centrifugalized  so  that  clear  serum  is  separated 
off.  The  clot  should  not  be  disturbed  too  energetically  as  it  is  best  to  get 
absolutely  clear  yellowish  serum  not  blood  tinged. 

The  following  mixtures  are  then  made  with  each  donor's  blood,  pref- 
erably within  12-24  hours  of  the  time  of  collecting  the  blood: 

(a)  3  parts  or  units  of  donor's  serum  and  i  part  or  unit  cf  recipient's  red 
cell  emulsion. 

(b)  3  parts  or  units  of  recipient's  serum  and  i  part  or  unit  of  donor's 
red  cell  emulsion. 

Controls: 

(c)  3  parts  or  units  of  donor's  serum  and  i  part  or  unit  of  donor's  red 
cell  emulsion. 

(d)  3  parts  or  units  of  recipient's  serum,  i  part  or  unit  of  recipient's 
cell  emulsion. 

(e)  3  parts  of  saline,  i  part  of  donor's  red  cells. 
(/)  3  parts  of  saline,  i  part  of  recipient's  red  cells. 

These  mixtures  are  made  in  very  small  test-tubes  (3X3/8  inch).     The. 


Il8  ,  .AGGLUTINATION 

quantity  comprised  by  each  "part"  or  "unit"  varies  according  to  the 
amount  of  blood  or  serum  obtained  from  each  donor  and  recipient.  If 
sufficient,  it  is  best  to  work  in  drops  (each  drop  being  about  0.05  c.cm.), 
thus  mixing  3  drops  of  serum  (0.15  c.cm.)  and  i  drop  of  the  red-cell  emulsion 
(0.05  c.cm.).  If  the  serum  is  not  sufficient,  a  smaller  arbitrary  unit  may 
be  used  in  the  form  of  Wright's  pipettes  (4  to  5  millimeters  caliber)  fitted 
with  rubber  nipples,  drawn  out  to  a  length  of  2  to  3  inches,  and  marked  off 
about  i  inch  from  the  tip  with  a  blue  pencil,  this  distance  comprising  the 
unit. 

The  tubes  are  placed  in  the  incubator  for  three  hours  and  then  in 
the  ice  box  for  24  hours.  Agglutination  when  it  occurs  does  so  rather 
promptly,  within  15  to  30  minutes  after  the  mixtures  have  been  made. 
It  is  recognized  macroscopically  by  the  clumping  of  the  red  blood  cells  into 
small  floccules  which  later  on  appears  like  a  distinct  clot.  A  hanging  drop 
preparation  of  such  a  mixture  shows  the  same  phenomenon;  usually  this  is 
unnecessary  as  the  macroscopical  appearance  is  characteristic.  If  one  is 
in  doubt,  however,  microscopical  examination  should  be  made.  Hemol- 
ysis,  if  pronounced,  is  observed  in  an  hour  or  even  less,  but  certainly  after 
the  three  hours'  incubation;  the  finer  grades  of  hemolysis  are  detected  after 
the  tubes  have  remained  in  the  ice  box  12  to  24  hours.  The  control  tubes 
must  show  no  agglutination  or  hemolysis. 

In  cases  where  for  any  reason  the  taking  of  blood  from  the  vein  is  not 
allowed  or  impossible  (as  in  infants),  Wright's  method  of  working  with 
small  quantities  (Epstein  and  Ottenberg)  should  be  resorted  to. 

The  skin  at  the  bed  of  the  finger  nail  or  of  the  lobe  of  the  ear  is  pricked 
deeply  with  a  Hagedorn  needle.  For  the  red  blood-cell  suspension  several 
drops  of  blood  are  allowed  to  flow  or  taken  up  with  a  dropper  and  expelled 
into  a  tube  with  sodium  citrate  solution.  The  red  blood  cells  are  washed 
and  diluted  as  before. 

For  the  serum,  three  or  four  Wright's  capsules  (see  under  opsonins)  are 
filled  with  blood  which  is  allowed  to  clot,  and  centrifugalized.  Each  cap- 
sule is  nicked  with  a  file,  allowing  the  capsule  to  be  broken  open  and  the 
serum  pipetted  off. 

For  making  the  mixtures  Wright's  pipettes  (four  to  five  millimeters 
caliber)  fitted  with  rubber  nipples  are  used.  With  a  blue  pencil  an  arbi- 
trary point  is  marked  off  upon  the  drawn  out  extremity  of  the  pipette. 
Three  volumes  of  serum  (a  bubble  of  air  between  each  volume),  and  one 
volume  of  cell  suspension  are  drawn  into  the  same  pipette.  These  ingre- 
dients are  mixed  by  running  them  gently  out  and  then  drawing  up  again 
into  the  pipette.  The  entire  mixture  is  then  drawn  into  the  body  of  the 
pipette  and  the  tip  is  sealed  in  a  flame.  Each  pipette  serves  as  a  little  tube. 

The  time  and  manner  of  incubation,  and  the  observation  for  hemolysis 
follow  the  rules  mentioned  above. 


TRANSFUSION  BLOOD   TESTS  1 19 

This  method  is  especially  valuable  when  a  very  great  number  of  donors 
are  to  be  examined.  If,  however,  a  sufficient  amount  of  serum  is  obtain- 
able the  editor  prefers  the  first  method  as  it  is  a  good  deal  simpler;  then, 
too,  the  total  amount  in  each  test  is  the  same,  thus  allowing  better  of 
comparison  as  to  the  intensity  of  hemolysis. 


CHAPTER  XI. 

PRECIPITINS. 

In  the  former  chapter,  the  phenomenon  of  agglutination  was  explained 
as  a  clumping  of  bacteria  occurring  when  serum  is  mixed  with  its  corre- 
sponding bacteria.  In  1897  R-  Kraus  described  a  phenomenon,  very 
closely  allied  to  the  one  just  mentioned.  He  found  that  when  an  immune 
serum,  for  example,  of  cholera,  typhoid,  or  pest,  is  mixed  with  the  clear, 
sterile  nitrate  of  the  respective  bouillon  cultures  of  their  bacteria  (instead 
of  the  bacteria  themselves),  the  clear  solution  becomes  turbid,  and  a 
precipitate  forms.  This  reaction  is  known  as  precipitation;  the  elements 
within  the  immune  serum,  precipitins;  while  the  substances  (antigen) 
with  which  the  precipitin  reacts  and  which  originally  stimulated  the  pro- 
duction of  the  precipitin,  precipitinogen. 

Like  all  biological  reactions,  the  phenomenon  of  precipitation  is  not 
limited  to  bacterial  immune  sera  and  culture  nitrates,  but  is  observed  when 
any  animal,  vegetable  or  bacterial  soluble  proteid  substance  is  mixed  with 
the  serum  of  an  animal  which  has  been  immunized  against  the  particular 
proteid  material  in  question. 

Tschistowitsch  and  Bordet  were  the  first  who  called  attention  to  these  non-bacterial 
precipitins.  Bordet  (1899)  found  that  the  blood  serum  of  rabbits  treated  with  the 
serum  of  chickens  gave  a  specific  precipitate  when  mixed  with  chicken  serum.  Tschi- 
stowitsch demonstrated  a  similar  reaction  with  the  sera  of  rabbits  treated  with  horse's 
and  eel  serum. 

The  biological  structure  of  the  precipitins  is  strongly  analogous  to  that 
of  agglutinins.  Many  authorities,  in  fact,  consider  them  identical.  What- 
ever has  been  said  in  regard  to  the  effects  of  heating  and  addition  of  acids 
or  alkalies  upon  agglutinins,  applies  equally  to  precipitins.  Moreover, 
they  also  are  composed  of  two  groups,  a  binding  (haptophore)  and  a 
functionally  active  (ergophore)  group.  If  the  latter  is  missing,  they  are 
known  as  precipitinoids,  and  can  interfere  with  precipitation  just  as  ag- 
glutinoids  do  with  agglutination. 

In  speaking  of  precipitation,  it  has  always  been  customary  to  differen- 
tiate between  bacterial  and  proteid.  For  practical  purposes  this  division 
is  superfluous  inasmuch  as  the  bacterial  precipitins  are  nothing  more  than 
precipitins  of  bacterial  proteids. 

120 


PRECIPITIN   REACTION  121 

Bacterial  Precipitin  Reactions. 

For  the  production  of  precipitating  sera,  animals,  preferably 
Production  of  rabbits,  are  injected  either  with  broth  cultures  or  salt  solution 
Bacterial      emulsions  of  agar  cultures  of  the  bacteria.     Five  to  six  in- 
Precipitat-    jections  of  gradually  increasing  quantities  are  given  intra- 
ing  Antisera.  peritoneally  or  intravenously,  at  intervals  of  from  five  to  six 
days.     The  dose  varies  with  the  virulence  of  the  bacteria. 
The  intravenous  method  frequently  giyes  the  stronger  serum  but  the  mode 
of  administration  depends  also  on  the  pathogenic  properties  of  the  micro- 
organism in  question.     The  immunized  animals  should  be  bled  about 
seven  to  twelve  days  after  the  last  injection  of  bacteria.     The  filtrates  of 
bouillon  cultures  and  the  various  forms  of  bacterial  extracts  will  also,  when 
injected,  produce  precipitins.     The  serum  from  individuals  undergoing 
an  infection,  or  convalescing  from  one,  contains  precipitating  bodies  against 
the  respective  infective  agent. 

Inasmuch  as  the  precipitin  reaction  consists  in  the  formation  of  a  pre- 
cipitate, it  is  important  that  both  of  the  ingredients  (precipitin  and  precipi- 
tinogen) be  absolutely  clear  and  have  no  tendency  to  spontaneously  become 
turbid,  or  form  a  precipitate. 

In  order  to  get  a  clear  serum  one  should  avoid  withdrawing 
Obtaining  the  blood  during  the  period  of  digestion  of  the  animal,  because 
Clear  Sera,  it  is  chylous  at  such  a  time.  In  man  the  best  occasion  for 

obtaining  the  blood  is  in  the  morning  before  breakfast.  As 
for  animals,  it  is  advisable  to  give  them  no  solid  food  (or  milk)  for  twenty- 
four  hours  previous  to  venesection.  Then  a  very  minute  quantity  of  blood 
is  withdrawn  and  immediately  centrifugalized  in  order  to  ascertain  whether 
the  serum  is  clear  or  not.  If  it  is  satisfactory,  larger  amounts  may  be 
collected.  The  presence  of  erythrocytes  and  bacteria  causes  a  serum  to  be 
turbid.  Simple  sedimentation  or  centrifugalization  suffices  to  overcome 
this. 

If  in  spite  of  these  precautions  turbidity  still  persists,  recourse  may  be 
had  to  filtration  through  paper  or  bacterial  filters,  preferably  new  ones. 
This  method  should,  however,  be  used  as  a  last  resort,  because  filtration 
always  tends  to  diminish  the  strength  of  a  serum. 

Bacterial  precipitinogens  are  prepared  by  filtration  either  of 

Bacterial     bouillon  cultures  or  bacterial  extracts.     The  filtrates  must  be 

Precipitin-    absolutely   clear;    also   sterile,    as  frequently  the  precipitin 

ogens.       reaction  requires  a  long  period  of  time.     If  bacteria  are  present 

they  may  grow  quickly,  and  produce  turbidity.  After  a 
time  the  precipitinogen  loses  its  property  of  combining  with  precipitins 
and  forming  precipitates.  In  such  a  case  the  precipitinogen  can  still  be 
employed  for  immunization  purposes. 


122 


PRECIP1TINS 


A  constant  amount  of  precipitinogen  is  placed  in  each  of  a  row 
Technique  of  °f  test- tubes,  and  to  these  are  added  diminishing  amounts  of 
the  Precipi-    the  immune  serum, 
tin  Reaction.   A  set  quantity  of  serum  and  varying  amounts  of  precipitinogen 

can  also  be  employed.  The  result  of  the  reaction  depends  to 
a  very  large  extent  upon  the  quantitative  relationship  of  these  in- 
gredients. //  relatively  too  much  precipitinogen  exists,  a  precipitate  will  not 
form.  An  already  formed  precipitate  will  dissolve  on  the  addition 
of  more  precipitinogen. 

The  explanation  of  this  peculiarity  is  unknown.  Since  colloidal  sub- 
stances, however,  at  times  give  similar  reactions,  many  authorities  have 
classed  the  precipitins  among  them. 

The  technique  of  a  precipitation  test  is  best  seen  in  the  following  table: 


Cholera 
bouillon 
filtrate 

Cholera 
serum 

Physiological 
saline  sol. 

Result  . 

After  4  hours 

After  24  hours 

5  .  o  c.cm. 
5.oc.cm. 

5  .  o  c  cm. 
5.0  c.cm. 
5.0  c.cm. 

i.o    c.cm. 
0.5    c.cm. 

o.  i    c.cm. 
0.05  c.cm. 

Very  cloudy. 
Cloudy. 

Faint  cloud. 
Clear. 
Clear. 
Clear. 
Clear. 
Clear. 
Clear. 

Clear;  marked  sediment  at  bottom. 
Clear  with  moderate  sediment   at 
bottom. 
Clear;  slight  sediment. 
Clear;  no  sediment. 
Clear;  no  sediment. 
Clear;  no  sediment. 
Clear;  no  sediment. 
Clear;  no  sediment. 
Clear;  no  sediment. 

0.5    c.cm. 

0.9    c.cm. 
0.95  c.cm. 
i.oo  c.cm. 
5.0    c.cm. 
0.5    c.cm. 
5.9    c.cm. 
5-  95  c.cm. 

i.o   c.cm. 
0.5    c.cm. 
o.i    c.cm. 
0.05  c.cm. 



A  parallel  row  of  tubes  with  normal  serum  should  be  included. 
If  highly  valent  sera,  such  as  are  obtained  by  immunization  with 
bacterial  extracts,  are  employed,  precipitation  may  result  soon  after 
mixing  the  two  constituents.  The  precipitins  are  strongly  specific,  al- 
though it  may  be  said  that  just  as  in  agglutination,  there  exists  in  precipita- 
tion a  certain  degree  of  "group  reactions." 

The   precipitation   test   has   no  clinical  diagnostic  value.     It 

Diagnostic     demonstrates  nothing  more  than  the  agglutination  test,  is 

Value  of       more  difficult  of  execution  and  associated  with  greater  sources 

Bacterial      of  error.     Only  occasionally  is  it  of  service  to  prove  the 

Precipitation,  presence  of  soluble  bacterial  substances  within  exudates  or 

organ  fluids. 

Porges  and  v.  Eisler  have  employed  the  precipitation  test  as  a  means  for  the  differ- 
entiation of  capsule-bacteria  where  the  method  of  agglutination  is  associated  with 
certain  difficulties.  The  precipitinogen  was  produced  by  filtration  of  four- weeks-old 


PRECIPITATION   TESTS   IN   TYPHOID   FEVER  123 

bouillon  cultures  of  pneumococci,  rhinoscleroma,  and  ozoena  bacilli.  The  immune 
serum  was  obtained  from  rabbits  which  had  received  four  to  five  subcutaneous  injec- 
tions of  the  respective  bacterial  suspensions. 

Fornet  has  recently  advocated  the  precipitation  test  as  an  aid  in  the 
clinical  diagnosis  of  typhoid  fever.  Although  his  attempts  have  not 
been  attended  with  practical  success,  the  principles  of  the  reaction  de- 
serve discussion  on  account  of  their  originality. 

Fornet  believed  that  it  should  be  possible  to  demonstrate  in  the  blood  of  typhoid 
patients  the  presence  of  the  antigens  (precipitinogens)  which  stimulate  the  antibodies, 
long  before  the  latter  themselves  become  evident.  He  actually  was  able  to  obtain 
turbid  mixtures  when  he  combined  precipitating  typhoid  serum  with  the  serum  of 
typhoid  patients.  In  many  cases  he  obtained  these  results  before  the  appearance  of 
the  Gruber-Widal  reaction. 

The  method  which  he  has  recently  employed  is  known  as  the  "ring 
test." 

Small  test-tubes  8  cm.  in  height  and  0.5  cm.  wide,  are  placed  in  rows  of 

Fornet's     twenty  each  in  a  small  black  test-tube  rack  so  arranged  by  the  help  of 

Ring  Test,   side  stands  that  the  tubes  are  inclined  at  an  angle  of  about  45°.    Across 

the  back  of  the  rack  is  attached  a  strip  of  dark  cloth  as  a  background  to 

facilitate  the  detection  of  any  precipitate.    The  immune  (or  convalescent)  serum  is 

placed  in  tubes   in   concentrated    and   diluted   form   1:5    and  1:10  with  normal 

saline,  and  then  the  serum  for  examination  in  concentrated  and  similar  dilutions  is 

carefully  floated  on  top  of  the  immune  serum.    The  mixtures  are  allowed  to  stand 

undisturbed  at  room  temperature  for  two  hours,  and  if  the  reaction  is  positive  a  whitish 

ring  at  the  point  of  contact  of  the  two  sera  makes  its  appearance.    A  control  test-tube 

of  normal  serum  plus  immune,  and  another  of  normal  plus  the  unknown  serum  in  the 

same  dilutions  as  those  employed  in  the  test,  must  remain  negative. 

Besides  in  typhoid  fever,  the  ring  test  is  also  evident  in  scarlet  fever,  measles  and 
syphilis. 

For  the  precipitation  test  in  syphilis,  the  serum  from  patients 

Syphilis      with  manifest  luetic  symptoms  is  employed  as  precipitinogen, 

Precipitation,  and  the  serum  from  individuals  with  general  paresis  acts  as 

precipitating  agent.  The  ring  test  must  be  carried  out  strictly 
in  accordance  with  the  rules  given  by  Fornet,  but  even  so,  its  diagnostic 
value  for  syphilis  is  still  doubtful.  Plaut  claims  that  normal  serum  gives 
the  reaction  just  as  often  as  luetic  serum;  this  is  strongly  denied  by  Fornet. 
Theoretically,  it  is  questionable  whether  these  precipitates  and  rings 
are  similar  in  origin  to  bacterial  precipitates,  or  whether  physical-chemical 
causes  are  at  the  bottom  of  the  former  phenomenon.  In  accordance  with 
the  latter  view  several  other  reactions  have  been  recently  recommended 
for  the  serum  diagnosis  of  syphilis. 

a.  Forges  and  Meier  noticed  that  luetic  sera  are  capable  of  producing 
Forges'      flocculent  precipitates  from  lecithin  solutions.     Forges  soon  found  the 
Reaction,    same  occurrence  with  solutions  of  bile  salts. 

Many  additions   and  modifications   have  been  made  to  the  Forges' 


124  PREC1PITINS 

reaction  since  it  was  first  recommended.     According  to  the  most  recent  publication, 
the  reaction  is  carried  out  as  follows: 
The  requirements  are: 

1.  One  per  cent,  solution  of  sodium  glycocholate  (Merck)  in  distilled  water. 

2.  The  patient's  serum  which  must  be  absolutely  clear,  and  heated  for  one-half  an 
hour  at  56°  C. 

Two-tenths  of  each  of  the  above  are  placed  into  a  narrow  test-tube  6  to  7  mm.  in 
diameter,  and  allowed  to  rest  for  sixteen  to  twenty  hours  at  room  temperature.  A 
positive  reaction  consists  of  the  appearance  of  distinct  coarse  flocculi,  which  as  a  rule, 
collect  near  the  surface.  Mere  turbidity  or  faint  precipitates  are  considered  as 
negative. 

The  original  Forges  method  of  employing  lecithin  was  not  at  all  specific,  the  reac- 
tion being  present  in  tuberculosis,  carcinoma,  and  other  infectious  diseases.  As  for 
the  new  modifications,  nothing  has  been  brought  forward  in  their  support. 

This  reaction  belongs  to  the  same  general  class  of  precipita- 
Klausner's  tion  tests  for  lues,  but  is  very  much  simpler  than  any  of  the 
Reaction,    others.     Two- tenths  c.cm.  of  absolutely  clear,  fresh  (at  the 
most,  two  hours  old),  active  serum  is  mixed  with  0.6  c.cm.  of 
distilled  water,  in  a  small  test-tube  7X0.5  cm.     Sera  containing  hemo- 
globin or  lipoids  are  not  suitable  for  this  reaction.     The  mixtures  are  al- 
lowed to  stand  at  room  temperature.     In  several  hours,  at  the  latest 
fifteen,  a  thick  flocculent  precipitate  2  to  4  mm.  high  appears  at  the 
bottom  of  the  tube.     Kreibich's  analysis  showed  it  to  consist  of  fibrin 
globulin. 

Apparently  this  substance  is  increased  in  luetic  serum  and  precipitated  by  the  dis- 
tilled water  in  which  it  is  insoluble.  Klausner's  reaction  is  by  no  means  specific  for 
syphilis  as  it  is  in  evidence  in  starvation,  typhoid  fever,  measles,  scarlet  fever,  pneu- 
monia, and  other  diseases,  as  well  as  during  health.  Nevertheless  it  must  be  said 
that  it  is  found  more  frequently,  earlier  and  much  stronger  in  lues  than  in  any  other 
condition. 

Klausner  states  that  in  fresh  cases  of  lues  the  best  reaction  is  seen  in  about  seven 
to  nine  hours,  while  in  older  cases  a  weak  reaction  appears  in  twelve  hours.  Mercury 
influences  the  test  in  that  the  interval  until  the  precipitate  becomes  marked,  is  pro- 
longed and  later  on  the  precipitate  becomes  fainter. 

In  spite  of  its  simplicity,  Klausner's  reaction  has  not  been  generally  adopted 
for  clinical  work,  as  the  Wassermann  reaction  with  its  far  greater  accuracy  has  re- 
placed it. 

Proteid  Precipitins. 

While  bacterial  precipitation  is  interesting  from  a  biological  standpoint 
but  bears  no  practical  significance,  proteid  precipitation  represents  one  of 
the  most  important  practical  aids  in  forensic  medicine.  By  this  means  the 
differentiation  of  various  proteids  can  be  easily  and  definitely  determined,  a 
problem  which  was  left  unsolved  by  chemistry. 

The  phenomenon  of  protein  precipitation  is  absolutely  analogous  to 


PRINCIPLES   OF   PROTEID   PRECIPITATION  125 

that  of  bacterial  precipitation.  If  a  clear  proteid  solution  (a)  is  mixed 
with  the  clear  serum  (a')  of  an  animal  immunized  against  the  above 
proteid  (a),  turbidity  and  precipitation  will  occur;  while  if  a  mixture  of 
the  serum  (a')  is  made  with  a  non-homologous  proteid  say  (b),  or  a  mixture 
of  the  proteid  (a)  with  the  serum  (b')  of  an  animal  immunized  against  b, 
no  precipitation  takes  place.  Graphically  expressed  it  looks  thus: — 

a-\-a'  =  precipitation. 
b-\-af  =  no  precipitation. 
a-\-bf  =  no  precipitation. 
b + b'  =  precipitation. 

In  other  words,  a  precipitating  immune  serum  reacts  only  with  its  homolo- 
gous proteid.  The  precipitin  reaction  is  specific. 

It  is  greatly  to  the  credit  of  Wassermann  and  his  co-workers 
Forensic  Use  ^'  Schiitze  and  Uhlenhuth,  who  recognized  that  this  speci- 
of  Albumin  n°ity  of  precipitins  was  of  great  medico-legal  value. 
Differentia- 
tion.       For  example,  a  bloody  shirt  is  found  in  the  home  of  a  man  charged  with 
murder;  the  prosecution  sees  in  that  the  proof  of  crime,  while  the  defend- 
ant pleads  that  the  stains  belong  to  the  blood  of  a  sheep;  the  proof  as  to 
their  source  is  of  the  utmost  deciding  evidence;  and  while  chemical  or  microscopical 
examinations  are  of  little  or  no  use,  serum  diagnosis  wins  the  day. 

The  blood-stained  clothing  is  extracted  in  water,  part  of  the  extract  is  mixed  with 
a,  the  serum  of  a  rabbit  immunized  against  human  serum  and  another  part  is  mixed 
with  b,  the  serum  of  a  rabbit  immunized  against  sheep's  serum.  If  the  mixture  a 
shows  a  precipitate,  it  can  be  definitely  stated  that  the  blood  stain  contained  serum 
derived  from  a  human  being;  while  if  mixture  a  is  clear  and  b  shows  the  precipitate, 
it  is  strongly  corroborative  of  the  presence  of  sheep's  serum. 

This  example  suffices  to  indicate  the  value  of  this  biological 
Blood      fact.     In  addition  the  reaction  is  made  use  of  in  the  deter- 
Relationship,  mination  of  the  nature  of  meats  (detection  of  horse  meat  sub- 
stitution for  beef). 

Furthermore,  this  method  has  explained  a  number  of  scientifi- 
cally interesting  problems.  Just  as  group  agglutination  demonstrated  the 
close  relationship  existing  between  various  bacteria,  so  also  serum  precipi- 
tation proves  a  distinct  relationship  between  the  different  species  of 
animals  (horse  and  donkey,  dog  and  fox,  hare  and  rabbit,  ape  and 
man,  etc.). 

Thus  the  serum  of  a  rabbit  immunized  against  human  serum  precipitates  not  only 
human  serum  but  also  that  of  monkeys;  the  serum  of  a  chicken  immunized  against 
rabbit's  serum  precipitates  not  only  that,  but  also  hare's  serum.  In  order,  however,  to 
differentiate  between  rabbit's  and  hare's  serum,  Uhlenhuth  advises  the  immunization 
of  a  rabbit  with  hare's  serum.  The  serum  of  such  an  immunized  rabbit  precipitates 


126  PRECIPITINS 

only  hare's  serum  and  not  rabbit's,  for  the  reason  that  "Isoprecipitins,"  i.e.,  precipitins 
against  the  same  kind  of  animal,  are,  as  a  general  rule,  not  developed.  Similarly  the 
differentiation  between  human  and  ape's  serum  can  be  accomplished  by  the  immuniza- 
tion of  apes  with  human  serum. 

Attempts  have  also  been  made  by  means  of  the  precipitation  reaction 
to  determine  the  origin  of  albumin  in  urine,  and  the  foreign  proteids 
circulating  in  the  blood  of  artificially  fed  infants. 

The  technique  remains  the  same,  independent  of  the  purpose  it  is 
employed  for.  It  consists  in  the  mixing  of  the  clear  precipitating  serum 
and  the  clear  proteid,  or  albumin  precipitinogen. 

Strongly  precipitating  antisera  against  proteid  solutions  are 
Production  prepared  by  methods  analogous  to  those  employed  for  the 
of  Proteid  production  of  antibacterial  sera.  The  use  of  rabbits  is 
Precipitating  generally  advised.  The  sera  or  proteid  solutions  should  be 
Antisera.  sterile.  Filtration  through  small  porcelain  niters  may  be 
necessary.  The  injections  may  be  made  subcutaneously, 
intraperitoneally,  or  intravenously.  The  subcutaneous  route  has  no 
advantage  unless  the  substances  to  be  used  are  contaminated;  but  then, 
larger  quantities  are  necessary.  The  intravenous  path  is  the  one  of 
choice.  A  single  injection  of  a  large  dose  (15  to  20  c.cm.),  or  three  injec- 
tions of  moderately  large  doses  (5  to  10  c.cm.)  on  three  successive  days 
may  be  given  and  the  animal  killed  after  7  to  10  days.  These  methods 
have  the  advantage  that  a  precipitating  serum  is  obtained  in  a  short 
period  of  time.  They  do  not  however  yield  as  strong  a  precipitating 
serum  as  the  following  slower  procedure.  One  c.cm.  of  the  solution  is 
injected  four  or  five  times  at  intervals  of  six  days.  The  dosage  may 
also  be  increased  at  each  successive  inoculation,  for  instance,  beginning 
with  2  c.cm.  of  an  animal  serum  and  increasing  gradually  through  3,  5 
and  8  c.cm.  to  possibly  15  c.cm.  at  the  last  injection.  The  animals 
should  be  weighed  from  time  to  time  and  if  considerable  loss  of  weight 
ensues  during  immunization,  the  intervals  between  injections  should  be 
increased. 

It  is  advisable  to  inject  five  or  six  animals  at  the  same  time,  and  by 
different  methods,  inasmuch  as  rabbits  vary  greatly  in  their  indi- 
vidual power  to  produce  precipitins  and  moreover,  because  some  die 
after  the  third  injection.  Frequently  only  one  serviceable  serum 
is  obtained,  even  though  the  immunization  of  five  rabbits  was  under- 
taken. 

Beginning  on  the  sixth  day  after  the  last  injection,  one  should,  at 
regular  intervals  of  one  or  two  days,  remove  a  small  quantity  of  blood  from 
the  vein  of  an  ear  and  test  the  strength  of  the  serum.  As  soon  as'  it  is 
found  to  be  satisfactory  the  animal  should  be  bled  and  its  serum  preserved 
on  ice,  with  precautions  for  sterility.  The  rules  given  above  for  obtaining 


127 

a  clear  serum  should  be  kept  in  mind.  If  the  precipitating  value  of  the 
serum  is  insufficient,  more  injections  may  be  given  before  the  animal 
is  finally  bled. 

If  the  serum  is  not  withdrawn  at  the  proper  time,  its  strength  begins 
to  diminish  and  further  injections  no  longer  stimulate  new  antibodies. 
It  is  even  possible  for  the  entire  precipitin  action  of  the  serum  to  dis- 
appear. 
Titration     ^^e  ^°^ow^nS  method  of  titration  of  the  strength  of  a  serum  is 

the  simplest.     One  c.cm.  of  various   dilutions  (i :  10,  i :  100, 

i :  1000,  i :  10000)  of  the  proteid  under  examination  (precipitin- 
ogen)  is  placed  into  different  test-tubes  and  o.i  c.cm.  of  the  precipitating 
serum  is  added  to  each.  The  tubes  should  not  be  shaken,  but  it  is  occa- 
sionally necessary  to  place  them  in  the  incubator  for  one  hour  before  any 
turbidity  or  precipitate  appears.  The  least  amount  of  proteid  solution 
which  still  distinctly  shows  a  precipitate  is  taken  as  the  titer  of  the  serum. 

For  medico-legal  purposes,  Uhlenhuth  advises  the  use  of  only 
Uhlenhuth's  highly  valent  sera. 

Method  of  He  considers  an  antiserum  as  efficient  if  o.i  c.cm.  of  it,  when 
Proteid  Dif-  mixed  with  its  respective  serum  in  the  dilution  of  i :  1000, 
ferentiation.  produces  a  distinct  turbidity,  either  at  once  or  in  one  to  two 

minutes  at  the  latest;  three  to  five  minutes  is  the  limit  for  an 
indication  of  turbidity  in  the  dilutions  of  i :  10000  and  i :  20000. 

Like  in  all  other  biological  reactions,  control  tests,  here  two  in  number, 
are  of  the  utmost  importance.  One  tube  must  contain  o.i  c.cm.  of  the 
precipitating  serum  mixed  with  i  c.cm.  of  saline,  another  o.i  c.cm.  of  the 
precipitating  serum  mixed  with  a  heterologous  serum  in  the  dilution  of 
i :  200  and  i :  1000.  Both  of  these  tubes  should  show  absolutely  no  pre- 
cipitate after  twenty  minutes.  In  this  way  the  specificity  of  the  pre- 
cipitin is  determined;  and  it  must  be  remembered  that  it  is  the  quantitative 
specificity  which  counts. 

In  the  process  of  the  determination  of  the  nature  of  meats,  it  is  es- 
pecially necessary  to  ascertain  exactly  the  precipitating  titer  against 
bovine  and  pig's  serum  possessed  by  the  rabbit's  precipitating  serum 
directed  against  horse's  serum . 

When  clear  solutions  are  at  hand  the  precipitin  reaction  is  compara- 
tively simple.  Frequently,  however,  the  test  must  be  performed  with  old 
and  dirty  blood  stains,  or  all  kinds  of  prepared  sausage,  so  that  the 
first  and  important  task  is  to  obtain  a  clear  solution. 

In  dealing  with  blood,  milk,  or  seminal  stains,  the  parts  of  the  clothing  involved  are 
excised,  divided  into  very  minute  shreds,  and  placed  in  a  test-tube  with  a  small 
amount  of  0.85  per  cent,  of  salt  solution.  If  the  material  is  not  too  old,  extraction 
for  one  hour  is  usually  sufficient,  otherwise  it  may  necessitate  a  period  of  twenty- 
four  hours  or  more.  Stains  upon  solid  material  such  as  steel,  wood,  stone,  etc., 


128 


PRECIPITINS 


are  carefully  scraped  off,  and  suspended  in  physiological  salt  solution.  To  obtain 
a  clear  solution  the  extract  must  be  passed  through  filter  paper  or  eventually  the  lilli- 
putian  bacterial  filter. 

In  the  examination  of  meats  or  other  food  stuffs,  it  is  best  to  remove 
the  material  for  examination  from  the  center  of  its  thickest  part,  as  this 
portion  has  been  least  exposed  to  the  methods  of  preservation,  especially 
the  high  temperatures.  Three  hours'  extraction  is  usually  sufficient;  the 
fresher  the  meat,  the  shorter  this  period.  Very  much  salted  meats  are 
best  washed  with  distilled  water,  previous  to  extraction.  Inasmuch  as  a 
great  deal  of  fat  interferes  with  the  reaction  it  is  advisable  to  remove  it 
beforehand  by  extraction  with  ether  and  chloroform  for  twenty-four 
hours  (Miessner  and  Herbst). 

Before  performing  the  actual  test  with  the  unknown  blood  stain,  it  is 
best  to  try  out  the  entire  reaction  with  a  similar  but  known  blood  stain  in 
order  to  make  sure  whether  all  the  ingredients  are  in  good  working  order. 
In  laboratories  equipped  for  medico-legal  examinations,  stains  made  upon 
linens  from  the  blood  of  man,  ox,  pig,  horse,  etc.,  are  always  kept  in 
readiness  for  such  preliminary  tests. 

Uhlenhuth  indicates  a  set  of  rules  to  be  observed  whenever  the  reaction 
is  undertaken.  They  are  here  cited  in  their  original  form,  as  practice  has 
shown  them  to  be  of  great  service. 

"In  order  to  obtain  sufficient  extract  for  the  test,  a  small  amount  of  the  material  is 
placed  in  a  test-tube  containing  5  c.cm.  of  normal  salt  solution.  This  must  not  be 
shaken.  After  one  to  two  hours,  2  c.cm.  are  poured  off  into  another  tube  and  gently 
shaken.  If  a  persisting  froth  appears  upon  the  surface  of  the  fluid,  it  can  be  taken  as 
proof  that  sufficient  extraction  has  occurred,  and  the  rest  of  the  fluid  is  thereupon  also 
transferred  to  this  tube.  If  no  froth  appears  the  2  c.cm.  should  be  returned  into  the 
first  test-tube  and  the  extraction  continued  until  repeated  tests  finally  show  the  pres- 
ence of  froth.  It  is  preferable  not  to  disturb  the  sediment  at  the  bottom  of  the  test- 
tube.  The  extract  eventually  obtained  may  have  to  be  filtered,  if  not  absoutely  clear. 

Such  an  extract  is,  as  a  rule,  stronger  than  that  required  for  the  test,  i.e.,  i  :  icoo. 
If  one  drop  of  a  25  per  cent,  nitric  acid  solution  is  added  to  i  c.cm.  of  a  i  :  1000  serum 
dilution  and  then  heated,  a  faint  opalescence  appears.  Enough  saline  should  there- 
fore be  added  to  the  final  extract  so  that  the  nitric  acid  test  corresponds  to  that 
given  by  a  dilution  of  i  :  1000. 

The  following  mixtures  are  then  made: 


Precipitating 

Normal 

Result 

Test  solution 

serum  from 
rabbit 

rabbit's 
serum 

saline 

After  five 
minutes 

After  twenty 
minutes 

i  c.cm.  i  :  1000 

O    I 

Opalescence. 

Turbidity   and  sedi- 

i c.c.m  i  :  1000 

O    I 

Clear. 

ment. 
Clear. 

O    I 

i  c.cm. 

Clear. 

Clear. 

PROTEID   PRECIPITIN   SPECIFICITY  1 29 

The  result  should  be  read  after  twenty  minutes  at  room  temperature. 
As  a  further  control  a  similar  row  of  tubes  should  be  made  with  the  extract 
of  the  non-bloody  part  of  the  clothing  in  order  to  show  that  the  latter 
alone  does  not  give  the  reaction. 

Even  putrid  or  otherwise  chemically  changed  proteids  may  still  give 
the  precipitin  reaction. 

The  precipitation  test  only  determines  the  animal  species  from  which 
the  proteid  originates,  but  cannot  prove  whether  it  comes  from  the  blood, 
semen,  milk  or  other  proteid  body.     In  order  therefore-  to  make  a  medico- 
legal  diagnosis  of  "  human  blood  stains,"  chemical  evidences 
"Origin"    must  in  addition  be  brought  forward,  that  the  stain  really 
and  "Con-  consists  of  blood.     Obermeyer  and  Pick  have  further  shown 
stitutional"  that  fcesides  animal  specificity   (" origin  specificity"),   pre- 
Specificity.  dpitation  also  demonstrates  the  "  constitutional  specificity  " 
of  proteids. 

If  instead  of  employing  pure  animal  or  plant  proleids  for  the 
immunization  of  animals,  variously  changed  albumins  are  used  (heated 
albumins,  acid  albumins,  formaldehyde  albumin,  etc.)  the  organism  reacts  by 
producing  antibodies  of  a  characteristic  nature,  different  from  those  de- 
veloped after  inoculation  with  the  pure  albumin.  For  example,  the  serum 
of  a  rabbit  imm  unized  for  a  long  time  with  horse's  serum  (normal  immune  pre- 
cipitin) will  produce  a  precipitate  when  mixed  in  vitro  with  the  pure  horse's 
serum  and  not  when  added  to  the  latter,  heated,  even  if  the  normal 
immune  serum  is  of  very  high  titer.  On  the  other  hand,  if  a  rabbit  is 
injected  with  horse's  serum  which  has  been  changed  by  being  diluted  and 
boiled  for  a  short  time,  the  immune  serum  thus  obtained  will  react  not  only 
with  normal  horse's  serum  but  also  with  heated  serum  and  a  group  of  its 
decomposition  products  with  which  the  normal  immune  serum  ordinarily 
never  induces  a  precipitate. 

This  fact  is  of  practical  application.  In  meat  substitution,  it  is  very  popular  to  boil 
the  sausage  in  order  to  make  detection  of  the  substituted  meats  more  difficult.  With 
the  aid,  however,  of  precipitins  produced  by  immunization  with  heated  proteids,  this 
fabrication  is  more  easily  detected  than  if  a  normal  immune  serum  were  used. 

•  While  animal  specificity  is  not  destroyed  when  the  albumins  are 
modified  in  the  above  manner  or  changed  by  tryptic  digestion  or  oxida- 
tion, Obermeyer  and  Pick  have  demonstrated  that  their  specificity  is 
lost  when  an  iodin,  nitro  or  diazo  group  is  inserted  in  the  proteid  mole- 
cule. Immunization  with  such  transformed  proteid  compounds,  e.g., 
xanthoprotein,  can  produce  a  precipitating  serum  which  will  react  with 
every  xanthoprotein  even  in  homologous  animals.  These  authors 
conclude  that  species  specificity  is  probably  dependent  upon  a  certain 
aromatic  group  of  the  proteid  molecule. 

9 


130  PRECIPITINS 

It  is  interesting  to  note  that  the  proteid  contained  in  the  lens  of  the  eye 
belongs  to  this  class  of  modified  proteids  which  possess  constitutional, 
but  no  species  specificity.  A  serum  produced  by  immunization  with  lens 
substance  will  react  with  the  proteid  derived  from  the  lens  of  any  animal, 
but  with  no  other  animal  proteid. 

In  conclusion,  the  origin  of  the  precipitate  formed  during  the 

Origin  of    precipitation  reaction  is  of  interest.     When  a  very  strong 

Precipitate,  precipitating   serum   is   employed,  the  precipitinogen  is   so 

greatly  diluted  that  it  no  longer  gives  any  of  the  chemical 
reactions  for  proteids,  but  nevertheless  yields  a  heavy  precipitate  when  the 
precipitating  serum  is  added.  This  surely  cannot  come  from  the  small 
trace  of  proteid  in  the  precipitinogen.  Furthermore,  if  the  immune 
serum  is  diluted,  the  formed  precipitate  becomes  comparatively  weaker 
and  disappears  entirely  if  dilution  is  increased.  It  is,  therefore,  generally 
considered  that  the  precipitate  originates  from  the  immune  serum. 


CHAPTER  XII. 

BACTERIOLYSINS  AND  HEMOLYSINS  (CYTOLYSINS). 

If  a  guinea-pig  is  immunized  with  living  or  dead  bacteria,  for  instance 
cholera  or  typhoid,  and  then  to  test  its  immunity  is  injected  with  a  single 
fatal  or  many  fatal  doses  of  living  bacilli,  the  animal  remains  alive; 
whereas  a  normal-control  animal,  not  treated  beforehand,  succumbs  to  a 
similar  inoculation.  In  order  to  determine  the  forces  to  which  the  im- 
munized animal  owes  its  protection,  Pfeiffer  undertook  the  following 
experiment:  Two  guinea-pigs,  one  immunized  and  another  normal,  were 
simultaneously  injected  intraperitoneally  with  living  cholera  vibrios,  and 
the  peritoneal  exudate  was  withdrawn  from  time  to  time  and  examined 
microscopically  in  hanging-drop  preparations.  (The  method  of  withdraw- 
ing the  peritoneal  fluid  with  capillary  pipettes  and  other  technical  details 
will  be  described  below.) 

A  very  striking  phenomenon  occurred.  While  the  cholera 
Pfeiffer's  vibrios  in  the  peritoneal  exudate  of  the  normal  animal  re- 
Phenomenon,  tained  their  form  and  motility  and  increased  in  number  con- 
tinuously until  the  animal  succumbed  to  the  infection,  the 
bacteria  in  the  peritoneal  exudate  of  the  immunized  animal  behaved 
quite  differently;  they  first  began  to  lose  their  power  of  locomotion,  then 
their  form  changed,  they  broke  up  into  evenly  small  shining  masses,  so- 
called  "granula,"  and  finally,  after  several  minutes  these  also  disappeared. 
Guinea-pigs  injected  with  the  peritoneal  exudate  from  these  infected 
immune  animals  remained  healthy,  and  nutrient  media  inoculated  with 
material  from  the  same  source  remained  sterile. 

The  above  experiment  is  named  after  its  discoverer,  Pfeiffer,  and  the 
phenomenon  itself,  "bacteriolysis." 

Bacteriolysis  is  a  strictly  specific  process.  If  an  animal  which  is 
immune  to  cholera  is  inoculated  with  typhoid  bacilli,  the  bacteria  markedly 
increase,  as  in  a  normal  animal.  The  process  by  which  this  bacteriolytic 
force  takes  place  is  clearly  demonstrated  when  a  mixture  of  living  cholera 
vibrios  and  blood  serum  of  a  guinea-pig,  which  has  been  actively  immunized 
against  cholera,  is  injected  into  the  peritoneal  cavity  of  a  normal  guinea- 
pig  and,  as  a  control,  normal  serum  mixed  with  living  cholera  vibrios  is 
inoculated  into  a  second  guinea-pig.  Here  the  exudates  on  examination 
from  time  to  time  show  that  in  the  peritoneal  cavity  of  the  animal  injected 
with  the  immune  cholera  serum,  the  same  phenomena  of  bacteriolysis 
occur  as  described  above,  leading  to  the  sterilization  of  the  peritoneal 
cavity,  and  protection  of  the  animal  from  illness.  In  the  control  animal , 

131 


132  BACTERIOLYSINS  AND  HEMOLYSINS 

however,  the  normal  serum  has  no  influence  upon  the  bacteria,  so  that 
they  increase  rapidly  and  kill  the  animal. 

It  is  evident  then,  that  the  bacteriolytic  power  resides  not  only  in  the 
actively  immunized  animal,  but  that  it  may  also  be  transmitted  to  other 
animals  by  means  of  the  former's  serum.  Bacteriolysis,  therefore,  is  not  a 
property  of  the  tissues  of  the  actively  immunized  animal,  but  is  to  be  traced 
to  specific  antibodies,  "Bacteriolysins"  which  circulate  in  the  blood 
serum  and  body  fluids. 

From  the  above  experiment  it  must  be  assumed  that  the  phenomenon 
of  bacteriolysis,  like  agglutination  and  precipitation,  can  be  demonstrated 
also  in  vitro.  The  earlier  investigations  in  this  connection,  however, 
were  unsuccessful.  Bordet  was  the  first  to  obtain  conclusive  results  and 
also  to  elucidate  the  cause  of  previous  failures. 

While  agglutination  in  vitro  and  bacteriolysis  in 'vivo  were 
Bacteriolysis  readily  produced  by  mixing  living  bacteria  with  old  immune 
in  Vitro,  serum,  bacteriolysis  in  vitro  did  not  occur  under  similar  cir- 
cumstances. But  when  freshly  drawn  blood  serum  or  exudate 
of  an  immune  animal  was  used,  bacteriolysis  took  place  in  vitro  also. 
(In  fact,  granule  formation  can  be  directly  observed  by  the  microscope.) 
When  the  serum  becomes  old — and  twenty-four  hours  is  sufficient  to 
cause  the  change,  it  loses  its  bacteriolytic  powers.  It  seems  at  first 
glance  as  if  bacteriolysins  may  be  active  outside  the  body  also,  but  that 
here  they  lead  only  an  ephemeral  existence.  This  view,  however,  is  not 
quite  correct;  for  " inactive"  serum,  which  has  become  "ineffective"  in 
vitro,  can  again  produce  bacteriolysis,  if  it  is  utilized  to  passively  immu- 
nize healthy  animals.  Something  must  exist  in  the  organism,  which 
supplements  the  inactive  bacteriolysins  and  restores  their  activity.  This 
"reactivating  substance"  is  independent  of  the  immunizing  process,  since 
it  is  to  be  found  in  normal  animals  also.  Furthermore,  inasmuch  as 
not  only  cholera  and  typhoid  immune  sera,  but  also  all  other  immune 
sera  and  not  only  guinea-pig's  serum  but  even  rabbit's,  horse's,  and  human 
serum  may  in  like  manner  be  reactivated,  it  is  evident  that  the  reactivat- 
ing agent  lacks  specificity.  On  account  of  this  peculiar  quality  of  supple- 
menting the  inactive  bacteriolytic  serum  so  that  it  can  develop  its  real 
effectiveness,  Ehrlich  called  the  reactivating  substance  "Complement." 
Accordingly,  the  complement  is  a  normal  non-specific  substance  which  is  found 
in  the  body  fluids  (particularly  abundant  in  the  blood  serum)  oj  every  organism; 
its  existence  is  evidenced  either  by  the  activation  or  reactivation  of  bacteriolytic 
antibodies. 

Bordet  demonstrated  that  the  apparent  ease  with  which  the  bac- 
teriolysins lose  their  activity  is  to  be  traced  not  to  these  bodies,  but  to 
the  complement.  If  a  small  amount  of  fresh  normal  serum  is  added  to 
bacteriolytic  serum  which  has  become  inactive,  reactivation  occurs  in 


PRINCIPLES   OF  BACTERIOLYSIS  133 

vitro,  that  is  to  say,  the  bacteriolytic  serum  regains  its  ability  to  dissolve 
bacteria.  The  bacteriolytic  power  of  fresh  immune  serum,  depends, 
therefore,  upon  the  fact  that  it  contains  not  only  bacteriolysins  but  also 
complement;  the  failure  of  old  immune  serum  to  produce  bacteriolysis 
is  accounted  for  by  the  lack  of  complement,  while  its  capacity  for  reac- 
tivation is  explained  by  the  still  present  bacteriolysins. 

As  the  above-described  experiments  indicate,  bacteriolysis  is  a  complex 
process,  which  is  produced  by  the  interaction  of  two  substances;  one,  the 
bacteriolysin,  is  formed  through  an  immunizing  process,  and  accordingly  is 
a  specific  antibody  of  great  stability,  while  the  other,  the  complement,  is  a 
normal  non-specific  and  very  labile  serum  substance. 

The  stability  of  the  immune  bacteriolysin  is  evident  in  its  resistance  to 
heat,  whereas  the  complement  is  thermolabile.  If  freshly  drawn  immune 
serum  is  heated  to  56°  C.  for  one-half  hour,  the  complement  is,  as  a 
rule,  rendered  ineffective,  while  the  bacteriolysin  is  not  in  any  way  in- 
jured; it  retains  its  specificity,  and  the  degree  of  its  affinity  to  antigen 
remains  unchanged.  Bacteriolysins  are  interfered  with  by  temperatures 
above  60°  C.  only. 

Concerning  the  finer  mechanism  of  bacteriolysis  there  are  two  opposing  views,  that 
of  Bordet  and  of  Ehrlich.  Without  considering  too  closely  the  remarkable  researches 
of  these  two  investigators,  the  synonyms  for  bacteriolytic  antibodies  usually  found  in 
the  literature  will  be  reviewed. 

In  attempting  an  explanation  for  bacteriolysis,  Bordet  has  recourse  to  certain  phe- 
nomena in  staining  technique.  There  are  some  substances  which  can  be  stained  only 
when  prepared  in  a  definite  way  by  means  of  another  substance,  a  so-called  mordant 
("Beize")  which  itself  is  not  a  stain.  According  to  Bordet,  the  specific  substance  pro- 
duced by  immunization  represents  a  kind  of  mordant  which  "sensitizes"  the  bacteria 
to  the  action  of  the  second  normal  non-specific  substance;  the  latter  is  really  the  active 
agent  in  causing  the  dissolution  of  bacteria  and  is  called  by  Bordet  "alexin"— an  older 
term  used  by  Buchner — in  contradistinction  to  "substance  sensibilitrice." 

Ehrlich,  on  the  other  hand,  advocates  a  more  chemical  conception  of  the  essential 
process  of  bacteriolysis.  He  believes  that  the  substance  formed  by  immunization,  which, 
for  the  sake  of  brevity,  is  called  the  immune  body,  is  characterized  primarily  by  the 
fact  that  it  has  two  binding  groups.  One  of  these  has  a  chemical  affinity  for  the  bac- 
terial cell  and  is,  therefore,  known  as  the  "cytophile  group,"  the  other  is  characterized 
by  its  binding  affinity  for  complement  and  is,  therefore,  known  as  the  "  complemento- 
phile"  group.  Also  because  of  its  two  binding  groups  (receptors)  the  immune  body 
itself  is  called  amboceptor,  that  is,  double  receptor. 

Thus,  according  to  Ehrlich,  bacteriolysis  takes  place  in  the  following  way :  The  cyto- 
phile group  of  the  amboceptor,  which  is  strictly  specific  for  its  antigen,  attaches  itself 
to  the  antigen,  for  instance  the  cholera  vibrio;  while  the  complementophile  group  binds 
the  complement.  The  complement  must  be  regarded  as  a  sort  of  digesting  (proteolytic) 
ferment.  Although  it  is  always  present  in  normal  serum,  it  is  not  effective,  because 
bacteria  have  no  affinity  for  it.  Only  through  the  medium  of  the  amboceptor  (Zwis- 
chen-Korper,  intermediary  body) ,  can  complement  bind  itself  to  bacteria  and  dissolve 
them. 


134 


BACTERIOLYS1NS  AND  HEMOLYSINS 


The  specificity  of  the  bacteriolytic  process  depends,  therefore,  on  the 
specificity  of  the  cytophile  group,  while  the  complementophile  group 
possesses  no  or,  strictly  speaking,  only  slight  specificity;  it  adapts  itself  to 
the  complements  of  very  many  though  not  quite  all  kinds  of  animals. 

Recent  experiments  have  proven  that  the  complement  consists  of 
two  different  parts,  the  middle  piece  and  the  end  piece. 

Technique  of  Bacteriolytic  Experiments. 

To  determine  the  occurrence  of  bacteriolysis  there  are  two  methods  of 
procedure  : 

1.  Pfeiffer's  experiment. 

2.  The  bactericidal  plate  method. 

I.  The  Pfeiffer's  Experiment. 

The  essentials  of  Pfeiffer's  experiment  have  been  described  at  the 
beginning  of  this  chapter.  Briefly,  it  consists  in  injecting  intraperito- 
neally,  in  a  normal  animal,  bacteriolytic  immune  serum  mixed  with  living 
bacteria.  The  resulting  bacteriolysis  is  studied  microscopically  by  with- 
drawing small  amounts  of  peritoneal  exudate  from  time  to  time.  If 
this  experiment  is  performed  with  various  dilutions  of  immune  serum,  and 
if  it  be  determined  at  what  dilution  bacteriolysis  fails  to  occur,  then  the 
bacteriolytic  titer  is  evident. 

The  details  can  best  be  understood  by  taking  a  practical  example.  It 
is  desired  to  find  the  bacteriolytic  titer  of  the  serum  of  a  patient  recovering 
from  typhoid  fever  by  means  cf  the  Pfeiffer  experiment. 

To  accomplish  this  task  the  following  ingredients  are  needed: 

1.  A  strain  of  bacillus  typhosus  of  known  virulence  for  guinea-pigs. 

2.  Patient's  serum,  sterile,  and  free  from  complement. 

3.  Guinea-pigs  of  250  gms.  weight. 

A  preliminary  experiment  must  be  performed  in  order  to  determine  the  virulence 
of  the  typhoid  strain. 

TESTING  THE -VIRULENCE  OF  STRAIN. 

.  _  , 

'• '     •  -'".••'  '• 
Guinea-pig  No.  i.   I  i/II  '09  One  loopful  of  a  typhoid  agar  culture  suspended  in  j  2/II  dead. 


i  UV.U1.  01  uuumoii,  mjecicu  incrapenioneaiiy. 

Guinea-pig  No.  2. 

i/II  '09  One-  half  loopfui  of  same 

2/II  dead. 

Guinea-pig  No.  3. 

i/II  '09  One-fifth  loopful  of  same  ....... 

2/II  dead. 

Guinea-pig  No.  4. 

i/II  '09  One-eighth  loopful  of  same                       , 

2/II  sick. 

4/II  dead. 

Guinea-pig  No.  5.  j  i/II  '09  One-tenth  loopful  of  same, 


2/II  sick. 
3/II  well. 


INCREASE    OF  VIRULENCE    OF  BACTERIA  135 

As  far  as  the  Pfeiffer  experiment  is  concerned  the  titer  of  virulence  in  this  case  is  1/5 
of  a  loopful  of  an  agar  culture  because  this  dose  is  fatal  within  twenty-four  hours.  In 
order,  however,  to  make  sure  of  excluding  all  individual  variations,  which  can  and 
occasionally  do  occur,  it  is  advisable  to  use  not  the  titer  dose,  but  its  fifth  or  tenth 
multiple,  that  is,  in  this  case,  one  loopful. 

Doses  larger  than  one  loopful  should  be  avoided,  so  that  if  any  par- 
ticular strain  of  typhoid  bacilli  is  not  sufficiently  virulent,  necessitating 
the  use  of  larger  doses,  the  virulence  must  first  of  all  be  increased.  This 
is  done  by  passing  the  organism  through  animals  such  as  guinea-pigs. 

The  method  is  as  follows:  A  very  large  dose  of  the  culture,  for  example 

To  Increase    the  surface  of  an  entire  agar  tube,  is  injected  intraperitoneally.     Every 

the  Virulence,  animal  succumbs  to  this  enormous  dose.     The  bacteria-laden  exudate 

from  the  abdominal  cavity,  which,  of  course,  must  be  removed  under 
sterile  precautions,  is  then  inoculated  into  a  second  guinea-pig  and  when  it  dies,  into  a 
third,  and  so  on.  As  a  rule,  after  passing  through  one  or  two  animals  the  bacterial 
strain  (which  must  be  grown  pure  from  the  cadaver)  becomes  more  virulent,  as  can  be 
proven  by  titration.  Very  often  the  virulence  is  increased  exclusively  for  the  species 
of  animal  used  and  occasionally  this  is  associated  with  a  decrease  in  virulence  for  other 
species.  After  a  series  of  passages  through  animals,  the  strain  reaches  a  maximum 
strength  beyond  which  it  cannot  be  increased.  The  degree  of  virulence  varies  with  the 
type  of  bacteria.  Typhoid  and  cholera  usually  reach  only  a  moderate  virulence  (i/io 
to  1/20  loopful);  the  bacteria  of  the  hog  cholera  group  can  acquire  a  distinctly  higher 
virulence;  for  instance,  B.  paratyphosus,  i/ioo  to  i/iooo  of  a  loopful,  while  the 
streptococcus  and  pneumococcus  reach  the  highest  figures,  1/10,000  to  1/1,000,000  of 
a  loopful. 

For  the  Pfeiffer's  experiment  with  cholera  or  typhoid,  the  most  suitable  strains  are 
those  of  such  a  virulence  that  1/5  to  i/io  of  a  loopful  injected  intraperitoneally  kills 
in  twenty-four  hours. 

The  serum  to  be  investigated  is  freed  of  its  complement  by 
Technique  of  heating  in  a  water-bath  for  one-half  hour  at  56°  C.  Then  a 
Pfeiffer's  series  of  dilutions  are  made  in  bouillon  (not  in  salt  solution) 
Experiments.  for  mstance  i/io,  i/ioo,  i/iooo,  etc.  A  c.cm.  of  each  dilu- 
tion is  put  into  a  test-tube  (a  sterile  pipette  should  be  used) 
and  rubbed  up  with  a  standard  loopful  of  an  1 8  to  24  hour  agar  culture 
of  typhoid  bacteria.  Finally  the  contents  of  each  test-tube  are  injected 
intraperitoneally  in  a  guinea-pig  of  250  gms.  weight. 

Inasmuch  as  small  amounts  are  apt  to  be  lost  when  aspirating  the  fluid 
with  the  syringe  as  well  as  when  pouring  the  bacterial  emulsion  into  a 
watch  glass,  it  is  better  to  rub  up  two  loops  of  the  culture  in  2  c.cm.  of 
bouillon  instead  of  i  loop  in  i  c.cm.,  and  then  withdraw  only  i  c.cm.  for 
use  in  the  experiment. 

The  following  controls  should  be  prepared: 

i.  Dilutions  of  the  serum  of  a  normal  person  (or  animal  of  the  same 
type)  +  typhoid  culture. 


136  BACTERIOLYSINS  AND  HEMOLYSINS 

2.  Dilutions  of  immune  serum  +  a  heterologous  culture. 

3.  (a)  Bouillon  +  typhoid-culture. 

(b)  Bouillon  +  heterologous  culture. 

The  study  of  the  bacteriolytic  phenomena  follows  the  inoculation. 
For  this  purpose  capillary  pipettes  to  withdraw  the  peritoneal  exudate  are 
prepared  according  to  the  directions  of  von  Issaeff. 

A  thin  glass  tube  is  heated  in  a  Bunsen  flame  almost  to  the  melting 
point,  then  removed  from  the  flame  and  immediately  drawn  out  with  a 
sudden  jerk.  Very  fine  capillary  pipettes  can  thus  be  made. 

The  removal  of  the  exudate  is  accomplished  as  follows:  a  small  cut  is 
made  with  scissors  through  the  skin  of  the  guinea-pig's  abdomen;  the 
capillary  pipette,  the  large  end  of  which  is  kept  closed  with  the  index 
finger,  is  forced  into  the  abdominal  cavity  with  a  single  push.  The 
pressure  of  the  finger  is  next  relaxed  and  the  tube  slowly  withdrawn. 
In  order  to  avoid  injuring  the  intestines,  the  precautions  usually  advised 
in  intraperitoneal  inoculations  should  be  observed  here.  The  author  has 
found  Friedberger's  method  of  holding  the  animal  very  serviceable  (see 
Fig.  5).  The  procedure  is  absolutely  painless,  moreover,  the  ordinarily 
sensitive  guinea-pigs  withstand  the  operation  almost  without  uttering  a 
sound. 

It  is  best  to  withdraw  the  exudate  immediately  after  the  injection  and 
then  at  intervals  of  five  to  ten,  twenty,  and  thirty  minutes,  etc.  Observa- 
tions are  made  directly  in  hanging-drop  preparations.  Stained  speci- 
mens are  less  reliable  and  instructive  because,  according  to  the  investiga- 
tions of  Radziewsky,  the  findings  are  dependent  upon  the  kind  of 
coloring  matter  used.  Bacteria  which  are  in  the  process  of  dissolution 
soon  lose  the  power  of  being  stained  by  methylene  blue,  while  they  retain 
their  affinity  for  carbol-fuchsin  and  aqueous  solution  of  gentian  violet. 
Granules  are  demonstrated  only  incompletely  in  stained  preparations. 

The  prognosis  for  the  animal  quoad  vitam  is  unfavorable,  if  bacterioly- 
sis does  not  occur;  good,  if  it  does.  Yet  there  are  exceptions  to  the  latter 
rule,  a  subject  to  which  reference  will  be  made  later  on.  Now  that  the 
most  important  technical  details  of  the  Pfeiffer  phenomenon  have  been 
considered,  the  following  protocol  will  more  clearly  illustrate  the  exact 
procedure. 

Titration  of  a  bacteriolytic  serum  (after  Pfeiffer). 


TITRATION   OF   A  BACTERIOLYTIC    SERUM 


137 


Guinea-pig  1-4-07 

One  loopful  of     -fo.i  typhoid     Beginning  of  bacterioly- 

Animal 

No.  i. 

typhoid  culture.            serum.              sis   after    10  minutes; 

remained 

^                                                                                                                   _, 

after  30  minutes  exu- 

alive. 

~^~ 

in  i  c.cm.  of  bouillon  intraperi- 

date  was  sterile. 

tone 

ally. 

\ 

Guinea-pig  1-4-07 

One  loopful  of 

H-O.OI  typhoid 

Beginning  of  bacterioly- 

Animal 

No.  2. 

typhoid  culture. 

serum. 

sis    after    15    minutes; 

remained 

c                    '                                    ^ 

after  20  minutes  only 

alive. 

*" 

in  i  c.cm.  .of  bouillon  intraperi- 

isolated,   non-motile 

toneally. 

bacteria,    many  gran- 

ules; after  40  minutes 

sterile. 

Guinea-pig  1-4-07 

One  loopful  of 

-f-o.ooi  typhoid 

Beginning  of  bacterioly- 

2/4   animal 

No.  3. 

typhoid  culture. 

serum. 

sis  after   15   minutes; 

slightly  ill. 

,^_                                                                                                                _, 

after  20  minutes  nu- 

3/4 animal 

^ 

in  i  c.cm.  of  bouillon  intraperi- 

merous    granules    and 

recovered 

toneally. 

also  many  non-motile      and    re- 

bacteria;  after  an  hour, 

mained 

no  bacteria  at  all. 

alive. 

Guinea-pig  1-4-07 

One  loopful  of 

+0.0001  typhoid 

After  20  minutes  gran- 

2/4 animal 

No.  4. 

typhoid  culture. 

serum. 

ules  and  many  motile 

found 

^                                                                                                                   t 

bacteria;  after  i  hour 

dead. 

in  i  c.cm.  of  bouillon  intraperi- 

motile    bacteria    very 

tone 

ally. 

numerous. 

Guinea-pig  1-4-07 

One-fifth  loopful 

After  20  minutes  gran- 

2/4   animal 

No.  5. 

in  i  c.cm.  of 

ules  and  many  motile 

found 

bouillon  intra- 

bacteria;  after  i  hour 

dead. 

peritoneally. 

motile    bacteria    very 

numerous. 

Guinea-pig  1-4-07 

One  loopful  of 

+o.  i  normal 

After  15  minutes  many 

2/4    animal 

No.  6. 

typhoid  culture. 

serum. 

granules,  also  isolated 

found 

motile  and  non-motile 

dead. 

^ 

in  i  c.cm.  of  bouillon. 

bacteria;  after  20  min- 

utes   motile    bacteria; 

after  30  minutes  motile 

bacteria  very  numer- 

ous. 

Guinea-pig  1-4-07 

One  loopful  of 

+0.1  typhoid 

Complete    similar  find- 

2/4 animal 

No.  7. 

bacteria,  para- 

serum. 

ings. 

found 

typhosus.  (Vir- 

dead. 

ulence  I/  10 

loopful.) 

in  i  c.cm.  of  bouillon. 

The  bacteriolytic  titer  of  the  tested  serum  in  this  case  would  lie  between  o.ooi 
c.cm.  and  o.oooi  c.cm.  and  could  be  exactly  determined  by  further  tests  which  would 
take  into  consideration  the  intermediate  doses. 


138  BACTERIOLYSINS  AND  HEMOLYSINS 

On  close  study  of  the  above  experiment,  it  will  be  noted  that  even  in 
those  cases  in  which  the  animals  died  of  the  infection,  bacteriolytic  phe- 
nomena were  not  altogether  absent.  They  occurred  particularly  in  the 
beginning  and  were  incomplete.  This  can  be  considered  as  evidence  of 
the  fact  that  even  normal  animals  possess  a  certain  supply  of  bacteriolysins 
which  are,  however,  readily  exhausted.  This  amount  of  normal  bac- 
teriolysin  in  serum  varies  greatly  with  the  species  of  animal;  thus  the 
sera  of  man  and  rabbit  contain  very  little  normal  bacteriolysins  for  cholera 
and  typhoid,  while  horse's  serum  is  well  supplied  with  the  same. 

According  to  Kolle,  a  loopful  of  virulent  cholera  vibrios  is  destroyed  in  the  peri- 
toneal cavity  of  a  guinea-pig,  by 

0.005  to  o.oi  c.cm.  of  normal  horse's  serum, 
o.oi    to  0.02  c.cm.  of  normal  ass  serum. 
0.02    to  0.03  c.cm.  of  normal  goat's  serum, 
o.i      to  0.3    c.cm.  of  normal  rabbit's  serum. 

The  protective  action  of  bacteriolytic  sera  differs  very  essentially  from 
that  of  antitoxic  sera.  For  the  latter,  the  law  of  multiple  proportions  holds 
true;  a  stronger  dose  of  toxin  is  neutralized  by  a  proportionately  larger 

amount  of  antitoxin;  to  bacteriolytic  sera  this  rule  does  not 
Endotoxin.  apply.     If  the  bacteria  are  increased  beyond  a  certain  quantity, 

their  dissolution  can  indeed  be  accomplished  by  the  addition  of 
sufficient  amounts  of  bacteriolysins,  but  the  animal  dies  nevertheless.  Its 
peritoneal  cavity  examined  during  life  or  postmortem  may  be  absolutely 
sterile.  Pfeiffer's  explanation  for  this  phenomenon  is  that  the  endotoxins 
within  the  bacteria  are  liberated  by  bacteriolysis  and  kill  the  animal. 
Fatal  results  from  endotoxin  follow  in  a  similar  manner  when  dead  instead 
of  living  bacteria  are  injected. 

Since  endotoxins  can  continue  their  effective  action  in  spite  of  the 
serum,  it  is  evident  that  the  usual  bacteriolytic  serum  lacks  the  power  to 
neutralize  the  poisons  of  the  endotoxins.  Many  investigators  have  at- 
tempted to  supply  this  deficiency.  (This  will  be  considered  later.) 

•  While  bacteriolysis  may  take  place  without  any  resulting  protective 
action,  on  the  other  hand  a  serum  may  be  curative  in  spite  of  the  absence 
of  bacteriolysis.  This  is  well  demonstrated  in  MetchnikofFs  experiment. 

A  marked  leucocytosis  in  the  abdominal  cavity  of  a  guinea-pig 

is  produced  by  the  intraperitoneal  injection  twelve  hours 
mkoff's  .  . 

Experiment    Prevlously  of  5  to  10  c.cm.  of  aleuronat  solution  or  sterile 

bouillon.  Pfeiffer's  experiment  is  then  performed.  As  a  rule, 
bacteriolysis  occurs  up  to  a  certain  point,  particularly  when  cholera 
vibrios  are  used;  most  of  the  bacteria,  however,  retain  their  form  and  are 
taken  up  by  the  leucocytes. 

Metchnikoff  used  this  experiment  to  uphold  his  theory  of  the  signifi- 


IDENTIFICATION    OF   BACTERIA  BY   PFEIFFER's    TEST  139 

cance  of  phagocytosis.  Pfeiffer  maintained  that  bacteriolysis  was  the 
most  important  protective  weapon  of  the  immune  organism,  against  bacte- 
rial invasion.  According  to  Metchnikoff  and  his  followers  among  whom 
Bail  in  particular  must  be  mentioned,  bacteriolysis  in  the  abdominal 
cavity  is  only  an  exceptional  phenomenon  (test-tube  experiment  in  vivo); 
its  occurrence  is  made  possible  by  the  circumstance  that  the  abdominal 
cavity  is  as  a  rule  almost  free  of  wandering  cells,  and  that  the  few  which  are 
present  are  so  injured  by  the  severity  of  the  infection  that  they  disinte- 
grate. If  their  number  increases,  bacteriolysis  does  not  occur,  or  at  least 
is  only  slight.  Likewise,  bacteriolysis  is  incomplete  in  the  presence  of 
cells,  for  instance  in  the  blood,  spleen,  liver  and  subcutaneous  tissue,  etc. 
A  detailed  consideration  of  this  much  mooted  problem  does  not  fall 
within  the  compass  of  this  book.  It  is  sufficient  to  have  pointed  out  the 
great  questions  of  fundamental  significance  which  hinge  upon  the  discus- 
sion of  the  Pfeiffer  experiment,  questions  which  concern  the  essential 
features  of  antibacterial  immunity.  It  can  be  readily  understood,  there- 
fore, why  the  phenomenon  of  bacteriolysis  has  been  so  much  studied, 
although  its  practical  significance  is  only  limited. 

The  Pfeiffer  experiment  can  be  used  for  the  differentiation  of 
The  Practi-  bacteria  as  well  as  for  the  demonstration  of  bacteriolysins  in 
cal  Applica-  serum.     It  serves  as  a  control  for  the  agglutination  reaction, 
tion  of  the 

Pfeiffer  Ex-  Pfeiffer  and  Kolle,  Brieger  and  others,  have  used  bacteriolysis  as  a 
periment.     method  of  estimating  the  immunity  obtained  by  active  protective  im- 
munization against  cholera  and  typhoid  in  man.     It  must,  however,  be 
questioned  whether  it  is  admissible  to  draw  conclusions  as  to  the  degree  of  active  im- 
munity from  the  height  of  the  bacteriolytic  titer  of  the  serum,  inasmuch  as  animals 
are  found  which  possess  no  active  immunity  and  still  have  sera  of  high  bacteriolytic 
powers. 

The  most  important  practical  use  of  the  Pfeiffer  experiment  lies  in  the 
identification  of  suspected  cholera  cultures.  In  Germany,  the  Pfeiffer  test 
made  with  the  vibrios  obtained  in  pure  culture  from  the  suspected  patients 
is  required  for  the  official  diagnosis  of  the  first  cases  of  cholera. 

The  serum  used  for  this  purpose  should  be  at  least  strong  enough  in 
amounts  of  0.0002  c.cm.  to  cause  the  disintegration  of  the  bacteria  in  one 
hour,  when  a  mixture  of  one  loopful  of  an  eighteen-hour  agar  culture  of 
cholera  in  i  c.cm.  of  nutrient  bouillon  is  injected  into  the  peritoneal 
cavity  of  a  guinea-pig. 

For  this  experiment  four  guinea-pigs  of  250  gms.  weight  are  used. 


140 


BACTERIOLYSINS  AND  HEMOLYSINS 


Animal 

Culture 

Serum 

Method  of 
injection 

Result  in  cholera 
cases 

No.  i  

One  loopful  of  an  1  8 

o.ooi  c.cm.  chol- 

Intraperitoneally. 

After  20  minutes  or 

hours'    growth    of 

era   serum=5 

at  the  latest  i  hour, 

culture     suspected 

times  the  titer 

bacteriolysis    oc- 

to be  cholera  in  i 

dose. 

curs;     animal    re- 

c.cm. of  bouillo'n. 

mains  alive. 

No.  2  

One  loopful  of  an  18 

0.002  c.cm.  chol- 

Intraperitoneally. 

After  20  minutes  or 

hours'    growth    of 

era    serum  =10 

at  the  latest  i  hour 

culture     suspected 

times  the  titer 

bacteriolysis    oc- 

to be  cholera  in  i 

dose. 

curs;     animal    re 

c.cm.  of  bouillon. 

mains  alive. 

No.  3  (con- 

One loopful  of  an  18 

o  01  c.cm.  of  nor- 

Intraperitoneally. 

Increase  in  number 

trol). 

hours'    growth    of 

mal  serum  =  50 

of  bacteria;  animal 

culture     suspected 

times  the  titer 

dies. 

to    be    cholera    in 

dose  of  the  im- 

i c.cm.  of  bouillon. 

mune  serum. 

No.  4  (con- 

One-fourth    loopful 

Intraperitoneally. 

Increase  in  number 

trol  of  vir- 

of   an     1  8    hours' 

of  bacteria,  animal 

ulence    of 

growth     suspected 

dies. 

culture)  . 

of  being  cholera  in 

i  c.cm.  of  bouillon. 

In  cases  of  subsiding  cholera,  the  Pf eiffer  experiment  is  performed  with 
the  serum  of  the  patient  in  dilutions  of  i  to  20,  i  to  100  and  i  to  500. 

Bacteriolysis  with  typhoid  organisms  is  less  typical  than  with  cholera. 
For  diagnostic  purposes  the  test  is  resorted  to,  only  when  the  agglutination 
reactions  are  doubtful.  When  bacteriolysis  also  gives  uncertain  results,  an 
animal  is  immunized  with  the  typhoid  suspected  bacteria  and  its  serum 
tested  for  its  power  of  agglutinating  or  destroying  definitely  known  typhoid 
bacteria  and  eventually  the  immunized  animal  may  be  injected  with 
virulent  typhoid  bacilli. 

Bacteriolysis  is  even  more  unsatisfactory  with  bacillus  paratyphosus 
B.  and  the  related  hog  cholera  group  of  organisms. 


While  with  typhoid  bacteria  the  onset  of  bacteriolysis  offers  a  favorable  prognosis  for 
the  animal,  guinea-pigs  inoculated  with  bacteria  of  the  paratyphoid-hog-cholera  group 
die  in  spite  of  complete  bacteriolysis.  Death  always  takes  place  late  (from  three  to 
six  days) ,  while  the  control  animals  succumb  in  about  twenty-four  hours.  Bacteriolysis 
has  also  been  observed  with  the  bacillus  of  dysentery  and  with  the  tubercle  bacillus; 
but  thus  far,  these  phenomena  have  gained  no  clinical  significance.  Bacteriolysis  does 
not  occur  in  anthrax,  pest  and  the  various  diseases  due  to  cocci. 


NEISSER   AND   WECHSBERG'S   BACTERICIDAL   TEST  141 

II.  Bactericidal  Plate -culture -method. 

(Plattenverfahren)  according  to  Neisser  and  Wechsberg. 

For  the  determination  of  the  bactericidal  titer  of  a  serum,  Neisser  and 
Wechsberg  recommended  the  so-called  bactericidal  plate-culture  method.  The 
principle  of  it  is  as  follows:  the  serum  to  be  tested  is  inactivated;  different 
amounts  of  this  inactivated  serum  are  mixed  with  a  definite  constant 
quantity  of  bacteria,  and  a  constant  quantity  of  active  normal  serum  is 
added  as  complement.  This  mixture  is  left  in  the  thermostat  sufficiently 
long  to  permit  the  occurrence  of  bacteriolysis.  Now,  to  determine  whether 
and  to  what  degree  death  of  bacteria  resulted  from  the  effect  of  the 
reactivated  bacteriolysins  (or  of  some  bactericidal  substance  otherwise 
unknown),  agar  is  added,  the  mixture  plated,  and  the  number  of  colonies 
counted. 

Stern  and  Korte  recommend  this  procedure  for  clinical  purposes,  as  a 
substitute  for  the  Pf  eiff  er  test  in  the  diagnosis  of  typhoid  fever.  They  point 
out  the  sparing  of  animals  as  one  of  its  advantages.  On  the  other  hand, 
this  method  consumes  much  more  time  and  its  results  are  less  trustworthy. 
It  has  not  found  a  place,  therefore,  in  clinical  practice. 

The  technique  of  Stern  and  Korte  is  the  following:  the  serum 
The  Tech-   °f  the  patient,  and  that  of  a  person  not  ill  with  typhoid  as 
nique  of     control,  are  inactivated  for  one-half  hour  at  56°  C.  and  i  c.cm. 
the  Method.  of  each  in  decreasing  dilutions  is  poured  into  sterile  test-tubes. 
To  each  is  added  0.5  c.cm.  of  a  twenty-four-hour  typhoid 
bouillon  culture  diluted  in  bouillon  to  i :  5000  or  i :  10000.     For  reactivation 
0.5  c.cm.  of  fresh  normal  rabbit's  serum  in  a  dilution  of  i  to  12  in  physio- 
logical saline  is  added  and  the  whole  thoroughly  shaken.     The  tubes  are 
then  placed  in  the  thermostat  for  three  hours.     The  entire  contents  of 
each  mixture  is  plated  on  agar,  and  after  eighteen  to  twenty-four  hours  the 
plates  are  to  be  examined.     That  particular  plate  is  considered  to  indicate 
the  extreme  limit  of  the  bacteriolytic  action  of  the  serum  in  which  there  is 
still  evident  a  very  great  decrease  in  the  number  of  colonies  as  compared 
with  the  innumerable  colonies  found  on  the  control  plates. 

Certain  other  controls  are  necessary: 

1.  One  tube  containing  culture  and  complement. 

2.  One  containing  culture  and  inactivated  immune  serum  in  the  highest 
concentration  used. 

3.  The  same  with  inactivated  normal  serum  instead  of  immune  serum. 

4.  Complement  without  culture  and  immune  serum  to  test  its  sterility. 

5.  Immune  serum  without  culture  and  complement  to  test  its  sterility. 

6.  One  tube  containing  only  culture,  to  be  plated  immediately. 

7.  One  tube,  containing  only  the  culture,  to  be  plated  after  standing  in 
the  thermostat  for  three  hours. 


142 


BACTERIOLYSINS   AND  HEMOLYSINS 


Topfer  and  Jaffe  pour  a  thin  layer  of  agar  into  a  petri  dish  and  let  it 
harden.  Upon  this  the  culture-serum- agar  mixture  is  poured,  and  after 
hardening  is  covered  with  another  thin  layer  of  agar.  In  this  way  the 
formation  of  a  film  of  culture  in  the  water  of  condensation  is  avoided. 

A  practical  example  is  appended  to  illustrate  the  plate  culture  method. 


Result  (poured  after  remaining 

3  hours  in  the  thermostat) 

Culture                          Serum 

Complement 

Normal  serum 

Immune  serum 

0.5  c.cm.  1/5000  typh. 

i/ioo        c.cm. 

0.5  c.cm. 

:  12  rabbit's. 

o  colonies. 

Many  thousand. 

0.5  c.cm.  1/5000  typh. 

1/500        c.cm. 

0.5  c.cm. 

:  12  rabbit's. 

100  colonies. 

Many  thousand. 

O.5  c.cm.  1/5000  typh. 

I/  1000      c.cm. 

0.5  c.cm. 

12  rabbit's. 

Many  thousand. 

Many  thousand. 

0.5  c.cm.  1/5000  typh. 

1/5000      c.cm. 

0.5  c.cm. 

12  rabbit's. 

00 

Many  thousand. 

O.5  c.cm.  1/5000  typh. 

i/ioooo    c.cm. 

0.5  c.cm. 

12  rabbit's. 

CO 

500 

0.5  c.cm.  1/5000  typh. 

1/20000    c.cm. 

0.5  c.cm. 

12  rabbit's. 

00 

200             . 

0.5  c.cm.  1/5000  typh. 

1/30000    c.cm. 

0.5  c.cm. 

12  rabbit's. 



0 

0.5  c.cm.  1/5000  typh. 

1/40000    c.cm. 

0.5  c.cm. 

12  rabbit's. 

5 

0.5  c.cm.  1/5000  typh. 

1/50000    c.cm. 

o.  5  c.cm. 

12  rabbit's. 

60 

0.5  c.cm.  1/5000  typh. 

i/iooooo  c.cm. 

o.  5  c.cm. 

12  rabbit's. 

800 

0.5  c.cm.  1/5000  typh. 

1/200000  c.cm. 

o  .  5  c.cm. 

12  rabbit's. 

CO 

Control  I 

0.5  c.cm. 

I  :  12  rabbit's. 

Many  thousand. 

0.5  c.cm.  i/soco  typh. 

Control  II  and  III       1  i/ioo        c.cm. 

— 

CO 

00 

0.5  c.cm.  1/5000  typh. 

Control  IV 

— 

0.5  c.cm. 

i  :  12  rabbit's. 

0 

Control  V 

i/ioo        c.cm. 

— 

0 

Control  VI 

— 

— 

Many  thousand. 

0.5  c.cm.  1/5000  typh. 

immediately  poured. 

Control  VII 

— 

— 

00 

0.5  c.cm.  1/5000  typh. 

poured  after  3  hours. 

'   ' 

In  addition  to  the  results  which  one  would  expect,  this  experiment  shows  one  strik- 
ing point.  With  the  normal  serum  the  tube  which  contains  the  largest  amount  of 
normal  bacteriolysins  shows,  on  plating,  the  fewest  germs.  The  greater  the  dilution  of 
the  serum  the  more  prolific  is  the  bacterial  growth.  The  titer  of  the  normal  serum  in 
this  case  lies  between  i/ioo  and  1/500.  The  controls  show  that  the  serum  and  comple- 
ment are  sterile,  and  that  the  inactive  normal  serum  is  ineffective.  During  the  three 
hours  in  the  thermostat  the  bacterial  suspension  has  become  stronger.  The  retarded 
growth  in  the  complement  culture  tube  can  be  traced  probably  to  the  presence  of 
normal  bacteriolysins. 

With  the  immune  serum,  on  the  other  hand,  results  are  quite  different.  Where 
the  most  concentrated  serum  is  used,  the  bacterial  growth  is  still  rather  profuse;  only 
the  moderate  doses  show  a  true  bactericidal  action  and  the  small  doses  are  altogether 
ineffective.  The  titer  of  this  serum  is  between  1/30,000  and  1/40,000. 


Neisser  and  Wechsberg  explain  this  phenomenon  by  the  so-called 
''deviation  of  the  complement."     They  assume  that  in  the  serum  of  higher 


DEVIATION   OF    COMPLEMENT 


143 


concentration  there  are  so  many  amboceptors  that  the  bacteria  cannot 
bind  them  all.  The  amboceptors  remaining  free  attach  themselves  to  the 
complement  by  means  of  their  complementophile  group  just  as  the  already 
bound  amboceptors  have  done.  Thus,  a  part  of  the  complement  is 
deviated  from  the  bacteria  and  only  an  incomplete  bacteriolysis  takes 
place. 

The  theory  of  complement  deviation  does  not  in  the  opinion  of  the  author  with- 
stand critical  examination.  Particularly  the  evidence  brought  forward  by  Bordet  and 
Gengou  that  the  affinity  of  complement  for  the  bacterium  -famboceptor  complex  (Sen- 
sitized bacterium)  is  considerably  greater  than  for  free  amboceptor,  militates  against 
the  view  of  Neisser  and  Wechsberg. 

It  is  possible  that  agglutination  may  account  for  the  phenomenon  of  deviation  of  the 
complement  in  that  the  agglutinated  masses  of  bacteria  afford  a  more  resistant  barrier 
to  the  action  of  the  bacteriolysins.  The  author  has  now  and  then  observed  an  analo- 
gous phenomenon  in  hemolytic  experiments;  strong  doses  of  hemolysin  were  less  effect- 
ive than  moderate  ones,  and  in  these  cases  the  momentary  hemagglutination  was 
readily  visible.  Also,  by  titrating  bactericidal  sera  in  animal  experiments,  it  has  been 
found  that  moderate  doses  often  afforded  the  greatest  protective  action. 

For  the  practical  application  of  the  plate  culture  method,  knowledge  of 
the  following  data  is  important,  as  it  is  necessary  to  consider  the  difference 
between  the  bactericidal  titer  of  sera  of  normal  and  of  typhoid  patients. 
According  to  Korte  and  Steinberg  the  bactericidal  titer  was 


Of  normal  cases 

Of  typhoid  cases 

Under  100  in  .  . 

74         per  cent 

o  o  per  cent 

Between  100  and  1000  in.  .  . 

8  6     per  cent. 

3  3  per  cent 

Between  1000  and  10  ooo  in. 

15  4     per  cent. 

15  i  per  cent 

Between  10,000  and  100,000  in  
Over  100,000  in  

2.0    per  cent, 
o  o    per  cent. 

23.3  per  cent. 
58  3  per  cent 

In  typhoid  fever  the  bactericidal  titer  does  not  run  strictly  parallel 
either  with  agglutination  or  the  Pfeiffer  experiments.  It  falls  toward  the 
end  of  the  disease  and  is  low  during  convalescence. 


Besides  its  use  in  typhoid  fever,  the  plate  culture  method  has  been  employed  for 
experimental  purposes  in  cholera  and  dysentery;  in  these  diseases,  however,  it  possesses 
no  clinical  diagnostic  significance. 

Concerning  bacillus  paratyphosus  B,  the  views  of  different  authorities  are  widely  at 
variance.  While  some  obtained  very  good  results,  similar  to  those  found  in  typhoid 
fever,  Topfer  and  Jaffe  could  demonstrate  no  bactericidal  power  whatever  in  vitro. 
This  difference  can  be  explained  only  by  variations  in  sera. 


144  BACTERIOLYSINS  AND  HEMOLYSINS 

Hemolysins. 

An  animal  that  is  injected  with  the  red  blood  cells  of  a  different  species 
develops  in  its  serum  antibodies  which  are  biologically  analogous  to 
bacteriolysins  and  differ  from  them  only  in  that  they  cause  disintegration 
of  erythrocytes  instead  of  bacteria.  These  antibodies  are  therefore 
called  hemolysins,  or  to  be  more  precise  immune-hemolysins,  since  they 
arise  through  a  process  of  immunization.  The  breaking  up  of  the  red 
blood  corpuscle,  hemolysis,  is  recognized  by  the  naked  eye.  The  hemo- 
globin passes  from  the  erythrocytes  into  the  surrounding  fluid  (serum  or 
physiological  salt  solution)  and  colors  it  red.  The  previously  opaque 
blood  lakes  and  becomes  transparent.  Immune-hemolysins  like  bac- 
teriolysins belong  to  the  class  of  amboceptors.  They  are  relatively  ther- 
mostabile  in  that  they  withstand  a  temperature  of  from  56°  to  58°  C. 
without  being  injured;  they  require  complement  for  the  develop- 
ment of  their  hemolytic  action.  Furthermore,  immune-hemolysins,  like 
all  amboceptors,  are  specific,  i.e.,  the  serum  of  a  rabbit  immunized 
against  horse's  blood  can  dissolve  only  the  blood  of  a  horse  and  not  that 
of  a  hen  or  cow.  On  the  other  hand,  group  reactions  occur  here  also;  for 
instance  the  immune-hemolysin  produced  in  a  rabbit  against  horse's 
blood  is  likewise  active  against  donkey's  blood. 

Just  as  various  antitoxins,  agglutinins,  precipitins  and  bac- 

Normal       teriolysins  can  be  found  in  normal  serum,  so  also  can  normal 

Hemolysin.    hemolysins  of  amboceptor  structure  be  discovered  in  the 

blood  of  many  animals. 

While  normal  hemolysins  come  into  play  in  only  a  few  reactions,  as  in 
several  modifications  of  the  Wassermann  test,  the  significance  of  immune- 
hemolysins  is  extraordinarily  great.  These  antibodies,  discovered  by 
Bordet,  and  independently  by  von  Dungern  and  Landsteiner,  were  care- 
fully studied  by  Ehrlich  and  Morgenroth  and  many  others.  Such  re- 
searches have,  first  of  all,  greatly  advanced  the  subject  of  immunity  in  its 
theoretical  aspects,  in  that  they  have  created  the  possibility  for  the  dis- 
covery in  minute  detail  the  finer  relationship  which  has  explained  some  of 
the  phenomena  occurring  in  bacteriolysis.  Furthermore,  the  studies  of 
hemolysins  led  to  the  discovery  of  the  complement  fixation  method,  a 
procedure  of  exceptional  practical  value. 

As  far  as  the  technique  for  obtaining  immune-hemolysins  is 

Production    concerned,  the  rules  which  hold  for  every  process  of  immu- 

ofHemo-     nization  are  naturally  to  be  followed  here  also.     It  is   not 

lytic  Sera,     possible,  however,  to  immunize  every  kind  of  animal  against 

every  type  of  red  blood  corpuscle.  Rabbits,  goats,  horses 
and  chickens  are  the  ones  which  are  best  adapted  to  supply  hemolytic  sera. 
An  animal  produces  a  better  hemolysin  the  remoter  its  relationship  to  the 


PREPARATION   OF   HEMOLYSIN  145 

animal  from  which  the  erythrocytes  for  injection  are  taken.  The  blood  to 
be  injected  can  be  employed  in  just  the  condition  in  which  it  flows  from 
the  vein.  Nevertheless  it  is  as  a  rule  defibrinated,  to  prevent  coagulation. 
The  simplest  and  most  practical  way  of  doing  this  is  to  place  some  glass 
beads  into  a  bottle  or  Erlenmeyer  flask  and  then  sterilize  it  by  dry  heat. 
The  blood  coming  from  the  vein  is  allowed  to  flow  into  one  of  these  flasks 
and  then  it  is  repeatedly  shaken  for  several  minutes.  This  suffices  to 
defibrinate  the  blood  and  thus  prevent  coagulation. 

The  production  of  hemolysins  depends  entirely  upon  the  red  blood 
corpuscles.  The  presence  of  the  serum  is  not  only  superfluous,  but  even 
harmful,  as  experience  has  shown  that  dangerous  reactions  may  follow 
the  injection  of  foreign  serum. 

Before  injecting,   therefore,   the   erythrocytes  are  washed.     For  this 
Washing  of  purpose  a  few  cubic  centimeters  of  defibrinated  blood  are  poured  into 
Red  Blood  a  centrifuge  tube  and  the  level  of  the  fluid  marked  on  the  tube.    An 
Corpuscles,  equal  or  double  this  amount  of  0.85  per  cent,  saline  is  added,  and  the 
tube  rapidly  centrifugalized.     The  erythrocytes  fall  to  the  bottom,  while 
the  upper  layers  of  the  tube  consist  of  diluted  serum  more  or  less  tinged  with  hemo- 
globin.    The  fluid  is  carefully  decanted,  fresh  saline  added,  the  tube  gently  shaken,  and 
again  centrifugalized.     If  this  is  done  two  to  three  times  the  erythrocytes  can  be  freed 
of  the  last  traces  of  serum;  finally,  by  adding  saline  up  to  the  mark  made  at  the  begin- 
ning of  the  experiment,  the  erythrocytes  are  obtained  in  the  normal  concentration, 
just  as  in  the  blood,  but  completely  free  of  serum. 

The  washed,  defibrinated  blood  can  be  injected  subcuta- 
Immuniza-  neously,  intravenously,  or  intraperitoneally.  With  the  sub- 
don,  cutaneous  and  intraperitoneal  methods  in  a  rabbit,  injections 

of  from  5  to  20  c.cm.  are  necessary  at  intervals  of  five  to  six 
days.  Far  larger  quantities  should  be  given  to  bigger  animals,  like 
goats  and  sheep.  Subcutaneous  injections  often  cause  infiltrations  and 
occasionally  abscesses.  The  author  therefore  uses  the  intravenous 
method  in  rabbits. 

A  suspension  of  washed  blood  corpuscles  is  diluted  four  to  five  times  with  physio- 
logical saline;  o.  5  to  i  .o  c.cm.  of  this  fluid  is  slowly  injected  into  the  ear  vein  every  five 
to  six  days.  Three  injections  are  almost  always  sufficient  for  procuring  a  good  serum. 
The  animals  sustain  the  first  two  injections  with  ease,  but  the  third  and  following  ones 
are  not  altogether  without  danger.  This  is  supposed  to  be  akin  to  anaphylactic  phe- 
nomena. It  is  therefore  advisable  to  immunize  several  animals  simultaneously,  so  that 
in  case  one  dies  there  is  another  to  replace  it.  Furthermore  there  are  such  marked 
individual  variations  in  the  ability  to  produce  hemolysins  that  it  is  best  to  have  several 
animals  to  choose  from.  Beginning  on  the  sixth  day  after  the  third  injection,  blood 
should  be  withdrawn  for  the  determination  of  the  hemolytic  strength  and  this  process 
repeated  daily  until  the  titer  has  reached  a  satisfactory  height  and  then  the  animal 
should  be  bled.  If  only  a  small  amount  of  hemolysin  is  needed,ithe  animal  can  be  allowed 
to  live;  it  will  gradually  lose  its  titer  completely  and  will  act  apparently  like  a  normal 


146  BACTERIOLYSINS  AND  HEMOLYSINS 

animal.  Nevertheless,  an  essential  difference  exists.  For  if  the  animal  previously 
immunized  is  again  injected,  hemolysins  reappear  after  a  short  incubation  period, 
whereas  in  a  normal  animal  a  prolonged  immunization  is  necessary.  Hemolysins, 
therefore,  exist  to  a  certain  extent  in  a  preformed  state  in  the  cells  of  an  immunized 
animal.  If  a  stimulus  to  immunization  occurs,  the  hemolytic  substances  are  thrown 
off  into  the  circulation,  while  in  a  normal  animal  the  formation  of  hemolysins  by  the 
cells  must  first  take  place. 

If  a  great  amount  of  hemolysin  of  the  same  titer  is  needed,  it  is 
The  Preser-  best  to  bleed  the  animal  to  death.     For  the  preservation  of 
vation  of    hemolysins  the  author  recommends  the  following  procedure 
Hemolysins.  which  he  has  found  very  trustworthy.     One  to  3  c.cm.  of 
serum  obtained  sterile  are  poured  into  sterile  tubes,  which 
are  closed  with  non-absorbent  cotton.     The  tubes  are  placed  into  a  water 
bath  at  56°  C.  for  one-half  hour  to  inactivate  the  serum  and  are  then  cov- 
ered with  sterile  rubber  caps.     (These  are  sterilized  by  placing  them  in  a 
i  per  cent,  sublimate  solution  for  forty-eight  hours.) 

An  immune  hemolysin  must  answer  both  qualitative  and  quantitative 
determinations;  qualitative,  whereby  is  proven  that  the  serum  can  hem- 
olyze  only  the  red  blood  cells  which  serve  as  antigen  or  to  a  slight  degree 
those  of  related  animals,  and  that  it  has  only  the  effect  of  a  normal 
serum  upon  the  erythrocytes  of  other  animals.  The  quantitative  estima- 
tion supplies  the  only  means  for  the  absolute  differentiation  between  a 
normal  and  an  immune  serum.  In  complement  fixation  where  hemolysis 
bears  an  active  part,  it  is  the  quantitative  use  of  the  hemolysin  which 
decides  the  result  of  the  reaction.  The  immune  serum  must  therefore 
be  "titrated." 

If  fresh  active  hemolytic  immune  serum  is  used,  a  constant  quantity  of 
blood  serving  as  antigen  is  mixed  with  decreasing  quantities  of  the  immune 
serum  and  the  mixtures  placed  in  the  thermostat.  Results  like  the  follow- 
ing will  be  obtained. 


Antigen  blood 


Hemolytic  serum  of  immune 
rabbit 


Result  after  2  hours 


i  c.cm.  of  5%  sheep's  blood !  i  c.cm.  of  active  serum,  i  to    10 

i  c.cm.  of  5%  sheep's  blood i  c.cm.  of  active  serum,  i  to    20 


i  c.cm.  of  5%  sheep's  blood, 
i  c.cm.  of  5%  sheep's  blood. 


i  c.cm.  of  active  serum,  i  to   50 
i  c.cm.  of  active  serum,  i  to  100 


Hemolysis. 

Incomplete  hemolysis. 
Incomplete  hemolysis. 
No  hemolysis. 


On  the  basis  of  this  experiment  the  titer  of  the  hemolytic  serum  for 
sheep's  blood  would  lie  between  i/io  and  1/20.  But  this  is  incorrect,  as 
it  was  pointed  out  previously  that  by  immunization  only  the  amboceptors 
are  increased  and  the  complement  remains  unchanged.  Each  of  the  above 


TITRATION    OF   IMMUNE   HEMOLYSIN  147 

dilutions  decreases  therefore  not  only  the  amount  of  hemolysin,  the 
quantitative  estimation  of  which  is  the  object  of  the  experiment,  but  also 
the  complement.  Inasmuch  as  the  latter  was  not  at  first  increased,  a 
point  is  soon  reached  where  there  is  no  complement  at  all  in  the  diluted 
fluid;  as  a  result  hemolysis  cannot  occur,  for  only  the  combination  of 
hemolysin  +  sufficient  complement  can  exhibit  any  hemolytic  action. 
Correct  titration  consists  therefore  in  allowing  varying  quantities  of  hemolysin 
with  a  constant  amount  of  complement  to  act  upon  a  constant  quantity  of 
red  blood  cells.  The  simplest  method  of  accomplishing  this  is  first  to  destroy 
the  complement  by  inactivation  of  the  hemolytic  serum,  then  to  make  the 
desired  dilutions,  and  finally  to  add,  to  all,  the  same  amount  of  normal  serum 
as  complement.  The  normal  serum  of  an  animal  of  the  same  species  as 
that  which  provided  the  immune  serum  can  under  no  circumstances 
serve  as  complement.  On  the  contrary,  foreign  sera  are  much  more 
suitable;  and  guinea-pig's  serum  is  especially  recommended  as  comple- 
ment when  immune  rabbit's  serum  is  used.  Not  every  complement  serves 
equally  well  for  any  immune  serum. 

A  very  good  hemolytic  system  and  one  which  is  almost  exclusively 
used  for  the  complement  fixation  reaction,  is  sheep's  blood  as  antigen, 
rabbit's  immune  hemolysin  as  amboceptor  and  normal  guinea-pig's  serum 
as  complement.  The  preparation  of  these  ingredients  should  be  carried 
out  as  follows: 

i.  Sheep's  Blood. — This  should  be  defibrinated  and  washed.  Washing 
is  necessary  because  fresh  sheep's  blood  contains  complement;  if  the 
blood  is  a  few  days  old,  washing  is  even  more  important. 

Although  serum  which  is  not  fresh  does  not  contain  sufficient  active 
Comple-  complement  to  cause  the  danger  of  superfluous  complement,  it  never- 
mentoids.  theless  contains  substances  which  interfere  with  hemolysis.  Probably 

the  existence  of  "  complementoids "  is  the  disturbing  factor.  It  must 
be  assumed  that  complement  is  composed  of  two  biologically  different  parts,  as  is  the 
case  with  toxins  and  ferments.  One  is  the  haptophore  group,  which  has  affinity  for 
the  complement ophile  group  of  the  amboceptor  and  is  the  more  stable  of  the  two.  The 
other  corresponds  to  the  energy  group  of  the  toxins  (toxophore  element)  and  of  the 
ferments.  Just  as  after  the  destruction  of  the  toxophore  group  there  remain  only 
innocuous  toxoids  whose  single  perceptible  activity  consists  in  their  ability  to  neutral- 
ize antitoxins,  so  also,  after  the  destruction  of  the  weakly  resistant  energy  elements  of 
the  complement,  there  remain  complementoids  which  lack  the  ability  to  activate  a 
bacteriolytic  o"r  hemolytic  amboceptor,  although  by  virtue  of  their  uninjured  hapto- 
phore groups  they  bind  the  complementophile  groups  of  the  amboceptors.  In  this  way 
they  usurp  the  place  of  whatever  active  complement  may  still  be  present,  rendering 
the  latter  inactive,  and  as  a  result  hemolysis  is  absent  or  incomplete. 

Following  the  technique  of  Ehrlich  and  Morgenroth,  a  5  per  cent,  sus- 
pension of  washed  red  blood  corpuscles  is  employed  to  test  a  hemolysin. 


148  BACTERIOLYSINS  AND  HEMOLYSINS 

A  pipette,  closed  at  the  top  by  pressure  of  the  index  finger,  is  thrust  to  the 
bottom  of  the  washed  erythrocytes  contained  in  the  centrifuge  tube;  a 
definite  amount,  for  instance  i  c.cm.,  is  withdrawn  and  allowed  to  flow 
into  a  graduate.  For  diluting  purposes  (in  this  case  up  to  20  c.cm.)  only 
isotonic  or  weakly  hypertonic  NaCl  solutions  may  be  used.  If  water, 
hypotonic  or  strongly  hypertonic  salt  solutions  are  employed,  the  red 
blood  cells  disintegrate.  This  is  not  a  true  biological  hemolysis,  but  de- 
pends upon  physical  basis.  0.85  per  cent,  saline  is  most  suitable  for  the 
majority  of  erythrocytes  (man,  rabbit,  guinea-pig,  ox,  sheep).  When, 
instead  of  an  isotonic  salt  solution,  an  isotonic  sugar  solution  is  made,  the 
red  cells  are  retained  in  their  proper  form,  but  the  addition  of  hemolysin 
and  complement  produces  no  hemolysis.  The  presence  of  salt  is  indis- 
pensable for  hemolysis  as  well  as  agglutination 

Undiluted,  unwashed,  defibrinated  blood  if  removed  sterile  can  be 
kept  several  days  in  the  ice-box.  The  "Frigo"  apparatus  is  unsuited 
for  this  purpose,  because  the  thawing  of  the  frozen  blood  breaks  the 
capsule  of  the  red  blood  corpuscle.  The  deterioration  of  the  preserved 
blood  is  recognized  by  the  large  hemoglobin  content  of  the  serum  or  the 
violet  color  of  the  blood. 

Occasionally  blood  left  in  an  ice-box  becomes  dark.  This  is  due  to  the  lack  of 
oxygen.  When  the  5  per  cent,  suspension  is  made  and  thoroughly  shaken,  the  red  color 
returns.  Such  blood  of  course  is  perfectly  serviceable. 

Still,  it  is  best  not  to  keep  blood  longer  than  four  days.  Blood  older  than  that,  even 
if  apparently  unchanged,  possesses  a  lowered  resistance  and  can  give  a  far  higher' titer 
in  hemolysin  tests  than  fresh  blood. 

2  The  rabbit's  hemolysin  must  have  been  inactivated  for  one-half 
hour  at  56°  C.  Dilutions  are  made  with  physiological  saline. 

3.  Guinea-pig's  complement  is  obtained  by  bleeding  to  death  a  healthy 
normal  animal. 

The  blood  is  allowed  to  flow  directly  into  a  centrifuge  tube  and  then  to  clot;  the  clear 
serum  is  obtained  by  centrifugalization.  For  titration  of  hemolysin  it  is  best  to  use  a 
constant  dose  of  complement  as  i  c.cm.  of  a  i/io  dilution.  Complement  can  be  kept 
for  twenty-four  hours  in  the  ice-box.  When  older  than  this  it  suffers  a  distinct  decrease 
in  efficiency  as  complementoid  is  produced.  (See  above.)  In  the  "Frigo,"  comple- 
ment may  be  kept  for  weeks.  Stern,  however,  does  not  recommend  complement  pre- 
served in  "Frigo"  for  use  in  complement  fixation  tests,  as  its  affinity  for  amboceptor 
is  noticeably  decreased. 

One  c.cm.  of  each  of  the  three  reagents  (each  so  diluted  with  saline  that 
the  desired  dose  is  contained  within  i  c.cm.)  is  mixed  and  2  c.cm.  of  0.85 
salt  solution  is  added  to  make  the  total  volume  up  to  5  c.cm. 

The  following  controls  are  absolutely  necessary. 


TITRATION   OF   HEMOLYSIN 


149 


1.  A  test  showing  that  hemolysin  in  strong  dosage  but  without  com- 
plement is  ineffective ; 

2.  A   test  indicating   that  without  hemolysin   complement   in   the 
dosage  used  is  ineffective; 

3.  A  test  which  shows  that  the  NaCl  solution  is  iso tonic. 

The  three  reagents  must  be  thoroughly  mixed  by  careful  shaking  of 
the  tubes  which  are  then  placed  in  the  thermostat  at  37°  C.  and  hemolysis 
watched  for.  The  duration  of  the  observation  is  a  matter  of  personal 
preference.  Only,  the  length  of  time  must  always  be  mentioned.  One 
must  say,  for  instance,  that  the  titer  of  this  hemolysin  is  i  :8oo,  using 
o.i  c.cm.  of  complement  under  observation  for  one-half  hour,  or  it  is 
i :  500  with  o. i  complement  under  observation  for  two  hours.  The  time  in 
which  hemolysins  work  is  very  different.  While  many  hemolysins  of  the 
same  titer  act  in  a  few  moments,  others  require  two  hours.  The  author 
has  made  it  a  rule  to  read  the  result  after  two  hours'  observation,  but  he 
notes  the  progress  of  the  reaction  every  one-half  hour  in  order  to 
determine  whether  it  is  a  slowly  or  rapidly  acting  hemolysin. 

The  following  chart  demonstrates  the  titration  of  a  hemolysin  as  a  preliminary 
experiment  to  the  complement  fixation  method. 


Result  of  hemolysis 

Antigen 

Amboceptor 

Comple- 
ment 

0.85  % 
Saline 

After  i  hr. 

After  i  hr. 

After  2  his. 

I. 

i  c.cm.  of  5  % 

i  c.cm.  dilution  1:10 

i  c.cm.  dilu- 

2 c.cm. 

complete 

complete 

complete 

sheep's  blood 

tion  i  :  10 

2. 

" 

I      100 

** 

41 

complete 

complete 

complete 

3- 

i( 

i     250 

11 

" 

complete 

complete 

complete 

M 

l( 

M 

,, 

Incom- 

Almost 

complete 

4- 

i    500 

plete 

complete 

5- 

11 

i     750 

" 

" 

Almost  o 

Incom- 

complete 

plete 

6. 

" 

I      IOOO 

" 

" 

0 

Incom- 

complete 

plete 

7- 

" 

I    1500 

" 

" 

0 

0 

Incom- 

plete 

8. 

I      2000 

o 

0 

o 

Control      I 

.< 

I      10 

3  c.cm. 

o 

o 

o 

Control    II 

.< 

i  c.cm.  dilu- 

o 

o 

o 

tion  i  :  10 

Control  III 

4  c.cm. 

o 

0 

0 

Determining  the  end  reaction  is  a  source  of  difficulty  for  the  beginner. 
Between  the  extreme  "o"  i.e.,  entire  absence  of  hemolysis,  where  the 
appearance  of  the  tube  corresponds  to  that  of  control  III  representing  a 
suspension  of  red  blood  cells  diluted  with  isotonic  saline,  and  the  other 
extreme  "complete/'  i.e.,  complete  hemolysis,  where  every  trace  of  cor- 
puscular elements  has  disappeared  and  a  fluid  looking  like  dilute  red-wine 


150  BACTERIOLYSINS  AND   HEMOLYSINS 

remains,  there  are  many  intermediate  stages.  These  intervening 
grades  of  reaction  are  represented  by  the  terms  almost  o,  incomplete, 
almost  complete,  and  similar  expressions.  The  meaning  of  the  terms  is 
self-evident.  How  any  particular  tube  is  to  be  designated  is  of  course  a 
subjective  question  since  the  so-called  transitional  stages  are  numerous. 

A  few  hours  after  the  reaction  is  ended,  a  remarkable  difference  may  be 
noted  between  the  tubes  in  which  hemolysis  has  occurred  and  those  in 
which  hemolysis  has  been  incomplete  or  totally  absent.  In  the  last  men- 
tioned, the  red  blood  cells  have  sunk  to  the  bottom  and  above  them  remains 
a  clear  fluid  which  consists  of  pure  saline  or  diluted  serum  (complement  + 
immune  serum)  and  is  colored  accordingly.  If  the  supernatant  fluid  is 
richer  in  hemoglobin  than  that  of  the  corresponding  control,  it  is  evident 
that  some  of  the  erythrocytes  were  hemolyzed  and  their  hemoglobin  set 
free.  If  the  erythrocytes  have  collected  at  the  bottom  apparently  in  the 
same  quantity  as  in  the  control  tube,  and  form  there  a  large  deposit,  a  trace 
of  hemolys  is  or  almost  o  would  be  the  terms  used  in  reporting  the  result. 
Tube  7  after  two  hours  showed  incomplete  hemolysis,  i.e.,  compared  with 
control  III  it  was  noticeably  clearer,  but  not  completely  transparent. 
After  twenty-four  hours  there  was  a  small  mass  of  undissolved  red  blood 
cells  at  the  bottom  of  the  test-tube  and  above  it  a  deep  red  fluid  which  was 
only  slightly  different  from  that  in  the  tubes  where  the  erythrocytes  were 
completely  dissolved.  If  this  sediment  should  become  so  small  that  on 
shaking  only  a  cloudy  turbidity  is  produced,  the  result  would  correspond 
to  the  designations  "very  small  sediment,"  "occasional  erythrocytes  at  the 
bottom  of  the  tube,"  or  "almost  complete  hemolysis." 

In  the  tubes  containing  inactive  hemolysin  without  complement  (control  I,  and  in 
complement  fixation  reactions)  hemagglutination  can  occur  because  the  agglutinins 
which  also  exist  in  the  serum  remain  active.  Hemagglutination  is  recognized  by  the 
shaking  the  sediment:  the  erythrocytes  are  not  equally  distributed,  but  remain  in 
clumps  or  strings  and  soon  sink  to  the  bottom  again. 

For  many  purposes  it  is  desirable  to  titrate  the  complement 

Titration  of   content  of  a  serum.     The  method  is  the  same  as  that  used 

the  Comple-   in  hemolysin  titration,  with  the  difference  that  a  fixed  amount 

ment.        of   hemolysin    and    varying    quantities  of    complement    are 

employed. 

The  titer  of  this  complement  when  employed  with  a  hemolysin  of  i/'iooo  strength 
and  allowed  to  stay  in  the  incubator  for  two  hours  would  be  0.04  c.cm. 

The  complement  content  of  the  serum  of  a  healthy  guinea-pig  is  fairly 
constant.  During  illness  the  titer  usually  is  decreased.  Among  healthy 
people  the  complement  titer  shows  marked  individual  variations. 

For  hemolysis  a  definite  quantitative  relationship  between  hemolysin 
and  complement  is  necessary. 


TITRATION    OF   COMPLEMENT 


Antigen 

Complement 

Result 

Amboceptor 

NaCl 

after    two 

solution 

hours 

i.    i  c.cm. 

5% 

of  sheep's  blood. 

c.cm.  hemolysin  dil.      :  1000. 

i  c.cm. 

:  10  (  = 

0.!). 

2.  o  c.cm. 

Complete. 

2.    i  c.cm 

f  G7 

of  sheep's  blood.  !      c.cm.  hemolysin  dil.      :  1000. 

0.8  c.cm. 

:  10   (  = 

0.08). 

2.2  c.cm. 

Complete. 

3.   i  c.cm. 

5% 

of  sheep's  blood.  .      c.cm.  hemolysin  dil.      :  1000. 

0.6  c.cm. 

:  10   (  = 

0.06). 

2.4  c.cm. 

Complete. 

4.   i  c.cm. 

5% 

of  sheep's  blood.        c.cm.  hemolysin  dil.      :  1000. 

0.4  c.cm. 

:  10   (  = 

0.04). 

2.6  c.cm. 

Complete. 

5.   i  c.cm. 

S  °7 

of  sheep's  blood.        c.cm.  hemolysin  dil.      :  1000. 

0.2  c.cm. 

:  10   (  = 

0.02). 

2.8  c.cm. 

Incomplete. 

6.   i  c.cm. 

5% 

of  sheep's  blood. 

i  c.cm.  hemolysin  dil.      :  1000.     i.o  c.cm. 

:  ioo(  = 

o.oi). 

2.0  c.cm. 

o 

7.   i  c.c.m 

5% 

of  sheep's  blood. 

i  c.cm.  hemolysin  dil.  i  :  1000. 



3.0  c.cm. 

o 

8.   i  c.cm. 

5  /o 

of  sheep's  blood. 

i  c.cm. 

i  :  10   (= 

=  0.1). 

3.0  c.cm. 

0 

9.   i  c.cm. 

5% 

of  sheep's  blood. 

ii 

4.0  c.cm. 

0 

On  the  basis  of  the  two  titrations  outlined  above,  it  is  estimated 
that  at  least  0.04  c.cm.  complement  is  necessary  to  activate  o.ooi  c.cm.  of 
hemolysin.  If  less  complement  is  used  with  the  same  amount  of  hemolysin, 
hemolysis  does  not  occur  or  else  it  is  incomplete.  If  the  quantity  of  hemol- 
ysin is  increased  for  instance  threefold,  then  it  will  be  found  that  0.02  c.cm. 
of  complement  suffices  to  produce  hemolysis.  Vice  versa  with  an  excess  of 
complement  the  hemolysin  titer  of  o.ooi  c.cm.  may  be  reduced.  How- 
ever, there  are  narrow  limits  to  this  mutual  compensatory  action. 

Cytotoxins,  Cytolysins. 

The  hemolysin  bodies  are  characteristic  and  important  members  of  a 
general  class  of  substances  known  as  cytotoxins,  especially  investigated  by 
Metchnikoff  and  his  co-workers. 

Just  as  the  immunization  with  erythrocytes  led  to  the  production  of 
lytic  amboceptors  which  in  connection  with  complement  destroyed  and 
dissolved  their  antigens,  so  in  a  similar  manner,  various  substances  more  or 
less  specific  for  their  antigens  have  been  produced  through  immunization 
with  leucocytes  "Leucocidin,"  with  nerve  tissue,  "neurotoxin,"  with 
spermatozoa,  "spermatoxin,"  and  kidney  tissue,  "nephro toxin."  The 
proof  of  their  action,  particularly  of  neuro-,  nephro-,  and  hepatotoxin  is 
not  simple.  As  all  these  cytotoxic  sera  have  at  the  same  time  a  hemolytic 
action,  it  is  not  easy  to  decide  to  what  extent  the  changes  in  the  organs 
observed  after  the  injection  of  the  cytotoxic  substances  are  dependent  upon 
the  action  of  hemolysins.  It  must  further  be  taken  into  consideration  that 
none  of  these  sera  are  absolutely  specific  for  the  organ  in  question.  This  is 
not  surprising,  inasmuch  as  there  are  widespread  common  group  charac- 
teristics (common  receptors)  among  the  different  organs  serving  as  antigens, 
and  only  very  few  groups  of  a  specific  nature.  The  hopes  which,  at  the 
beginning,  were  placed  upon  the  study  of  cytotoxins  particularly  with  the 
expectation  that  they  would  tend  to  become  diagnostic  and  therapeutic 
methods  for  the  treatment  of  malignant  tumors,  have  as  yet  been  unreal- 
ized. The  entire  field  of  cytotoxins  urgently  requires  further  investigation. 


CHAPTER  XIII. 

THE  METHOD  OF  COMPLEMENT  FIXATION. 

Its  principle.     Antituberculin.     Ehrlich's  side-chain  theory.     Serum  diagnosis  of 
syphilis,  and  diseases  caused  by  animal  parasites. 

It  has  already  been  demonstrated  that  neither  bacteriolysis 

The  Question  nor  hemolysis  can  take  place  without  the  presence  of  comple- 

of  Multipli-  ment.     The  question  therefore  arises  whether  this  complement 

city  of  Com-  is  the  same  in  both  of  these  reactions  or  whether  normal  serum 

plements.    possesses  different  complements.     In  order  to  solve  this,  a 

number  of  very  complicated  experiments  have  been  carried 
out  by  Ehrlich  and  Morgenroth,  Metchnikoff,  and  Bordet  and  Gengou. 
Ehrlich  and  Morgenroth  endeavored  to  show  that  not  only  do  the  comple- 
ments of  different  animals  of  the  same  class  vary,  but  that  numerous  com- 
plements exist  within  one  individual  serum  (conception  of  the  multiplicity 
of  complements).  Metchnikoff  believed  that  each  serum  contained  at 
least  two  complements,  the  microcytase  and  the  macrocytase,  thus  enlist- 
ing the  supporters  of  a  dualistic  theory.  Bordet  and  his  school,  on  the 
other  hand,  although  agreeing  with  the  idea  that  the  complement  varies  in 
different  animals,  deny  its  multiplicity  and  contend  that  any  given  serum 
contains  but  one  alexin,  or  complement — the  theory  of  unity  of  complement. 
It  would  be  superfluous  to  cite  all  the  experimental  data  supporting  these 
opinions,  but  nevertheless  a  review  of  the  classical  experiment  of  Bordet 
and  Gengou  which  corroborated  the  existence  of  only  one  complement,  thus 
offering  the  fundamental  principle  for  the  establishment  of  the  most  impor- 
tant method  of  serum  diagnosis,  namely,  complement  fixation,  would  not 
be  out  of  place. 

Bordet  and  Gengou  mixed  in  a  test-tube  typhoid  bacteria 

The  Principles  (antigen),  inactivated  typhoid  immune  serum  (amboceptor) 

^enTrfcT    anc*  norma^  semm  (complement).     Union  of  the  bacteria  and 

ation.        immune  serm  first  took  place  followed  by  absorption  of,  and 

coalescence  with,  the  bacteriolytic  complement  contained  in 
the  normal  serum.  As  a  result,  bacteriolysis  occurred  and  the  bacteriolytic 
complement  was  used  up  during  this  process.  Bordet  and  Gengou  rea- 
soned that  if  the  bacteriolytic  and  hemolytic  complements  were  identical, 
then  in  the  above  mixture  of  typhoid  bacteria,  immune  serum  and  normal 
serum,  the  hemolytic  as  well  as  bacteriolytic  complement  should  be  absent, 
while  if  the  plurality  of  complement  exists,  the  hemolytic  complement 
should  still  be  present.  Accordingly,  after  a  certain  interval,  washed 
erythrocytes  and  inactivated  homologous  immune  serum  were  added  and 

152 


BORDET   AND   GENGOU  PHENOMENON  153 

hemolysis  looked  for.  No  hemolysis  took  place,  thereby  attesting  to  the 
fact  that  the  bacteria  in  the  first  part  of  the  test  had  "fixed"  ("held  in 
check")  not  only  the  bacteriolytic  but  also  the  hemolytic  complement. 
Bordet  and  Gengou  thereupon  named  this  test  "complement  fixation"  or 
"complement  binding"  (La  fixation  d'alexine). 

With  the  aid  of  this  experiment  Bordet  and  Gengou  were  able  to  prove  a  number  of 
theoretically  important  points.  They  demonstrated  that  absorption  of  complement 
was  not  necessarily  accompanied  by  bacteriolysis.  For  example,  the  anthrax  and 
pest  bacteria  when  mixed  with  their  respective  homologous  immune  sera  show  no 
or  only  very  incomplete  bacteriolysis.  The  erroneous  conclusion  thus  reached,  to 
the  effect  that  these  sera  contained  no  amboceptors,  was  disproved  by  Bordet  and 
Gengou,  who  demonstrated  that,  i,  these  sera  contained  amboceptors  in  spite  of  the 
absence  of  bacteriolysis,  2,  the  complement  was  absorbed,  although  no  bacteriolysis 
took  place. 

During  the  process  of  immunization,  amboceptors  were  found  far  more  frequently 
than  bacteriolysins.  These  two  terms  must  not  be  considered  as  synonymous. 

Amboceptor  signifies  a  more  generic  term,  and  one  must  differentiate  between  amboceptors 
of  cytolytic  and  non-lytic  properties.  Whether  the  difference  here  really  depends  upon 
the  different  nature  of  the  amboceptor,  or  upon  the  construction  and  constitution  of 
the  antigen,  is  not  solved. 

The  fixation  of  the  complement  precedes  the  act  of  bacteriolysis.  The  important 
requirement  for  the  fixation  is  an  antigen  which  has  been  sensitized  by  the  attachment 
of  the  amboceptor*  thus  increasing  the  affinity  toward  the  haptophore  group  of  the 
complement.  Antigen  alone,  or  even  amboceptor  alone,  binds  the  complement  only 
very  slightly  or  not  at  all.  Whether  the  zymotoxic  (energy)  group  of  the  comple- 
ment manifests  its  activity  (bacteriolysis)  or  not  (absence  of  bacteriolysis)  is  materially 
indifferent  for  the  complement  fixation. 

Through  complement  fixation,  as  introduced  by  Bordet  and 
Complement  Gengou,  one  is  enabled  to  prove  the  presence  of  specific  anti- 
Fixation  as  bodies  when  the  antigen  is  known  or  reversely,  an  unknown 
a  Method    antigen  provided  the  specific  antibody  is  given.     This  method 
of  Serum    of  serum  diagnosis  can  be  widely  employed,  as  the  majority  of 
Diagnosis.    Dac^eria  an(j  immune  sera  (with  the  exception  of  pure  antitoxic 
sera)1  when  mixed  homologously,  give  a  positive  reaction — 
the  absence  of  hemolysis,  proving  the  absorption  of  complement  by  the 
union  of  the  antigen  and  its  specific  amboceptor.     This  reaction  is  strongly 
specific.     If  bacteria  are  mixed  with  an  inactive  heterologous  immune 
serum,  or  with  a  heated  normal  one,  not  in  concentrated  form  (normal 
amboceptor),  and  complement  is  added,  the  latter  will  not  be  fixed  but 
remains  to  be  taken  up  by  the  subsequently  added  red  blood  cells,  and  its 
immune  serum,  causing  hemolysis.     Hemolysis  indicates  that  the  mixed 
bacteria  and  serum  are  not  homologous,  as  the  complement  is  left  free,  and 
given  a  chance  to  unite  with  the  added  erythrocytes  and  hemolytic  ambo- 
ceptor.    In  the  case  where  the  bacteria  are  known,  e.g.,  typhoid  bacilli, 
the  occurrence  of  hemolysis  indicates  that  the  examined  serum  contains  no 

1  Even  antitoxic  sera  are  said  by  Nicolle  to  give  complement  fixation  reactions. 


154 


METHOD   OF   COMPLEMENT  FIXATION 


typhoid  amboceptors.  If  the  serum  is  known  (e.g.,  meningococcus  serum) 
the  occurrence  of  hemolysis  proves  that  the  bacteria  under  examination 
are  not  meningococci.  The  absence  of  hemolysis  will  in  the  first  case 
point  out  that  the  unknown  serum  contains  typhoid  amboceptors,  i.e.,  is 
a  typhoid  serum;  while  in  the  second  case  the  absence  of  hemolysis  would 
bear  definite  evidence  in  favor  of  meningococci.  The  accompanying 
figures,  15  and  16,  represent  schematically  the  positive  and  negative 
complement  fixation  test. 


Typhoid  bacilli  (antigen) 

Typhoid 

Inactive  typhoid  serum  (typhoid  am- 
boceptor) 

I  Typhoid 

Complement  amboceptor 


Hemolysin  (hemolytic  amboceptor) 

Sheep's  red  blood  cells  (2d  antigen) 
Result:     Due   to  the  union  of  comple-  Complement 
ment  with  the  complex  typhoid  bacillus 
-f-  the   typhoid  amboceptor,  hemolysis 
does  not  take  place. 

FIG.  15. 


p 

V7      Blood  cell 

V 

Hemolytic 

A 

amboceptor 

A 

Typhoid 
bacilli 


Typhoid  bacilli  (antigen) 

Inactive  cholera  serum  (cholera  ambo- 
ceptor) 

Complement 

I  Cholera 

Hemolysin  amboceptor 

Sheep's  red  blood  cells 
Result:    The   complement  unites  with 
the  hemolysin  and  the  sheep's  red  blood 
cells  thus  producing  hemolysis. 

FIG.  16. 


V7  Red  blood  cell 

VI 

Hemolytic 
amboceptor 


Complement 


Gengou  further  showed  that  not  only  cellular  antigens  can  stimulate 
the  formation  of  amboceptors,  but  that  during  the  course  of  immunization 
with  proteids  in  solution  (milk,  serum,  etc.),  complement  binding  ambo- 
ceptors are  also  formed  in  addition  to  the  precipitins.  Citron  has  there- 
fore proposed  the  term  "  antigenophile,"  to  designate  the  "cytophile" 
group  of  the  amboceptor. 

Widal  and  Lesourd  were  the  first  to  make  practical  application  of  the 
complement  fixation  property.  They  found  that  the  Bordet-Gengou  re- 
action could  be  obtained  far  more  frequently  and  earlier  with  the  serum  of 
typhoid  patients  than  the  agglutination  test.  Nevertheless,  this  entire 
complement  fixation  method  remained  unheeded  for  several  years. 


PRACTICAL   APPLICATIONS    OF   THE    COMPLEMENT-FIXATION   TEST      155 

Moreschi  (at  Pfeiffer's  institute),  while  conducting  some  theoretical  studies  con- 
cerning the  nature  of  anticomplements,  i.e.,  such  substances  which  tend  to  neutralize 
the  action  of  complements,  discovered  anew,  that  by  the  mixture  of  a  soluble  proteid 
with  its  antiproteid  serum  the  existing  complement  disappeared.  This,  as  has  been 
seen,  can  be  explained  by  the  presence  within  the  immune  serum  of  bodies  similar  to 
Gengou's  amboceptors.  Moreschi,  however,  stated  that  the  complement  disappeared 
because  it  was  thrown  to  the  bottom  mechanically,  by  the  occurrence  of  precipitation. 
Such  a  physical  explanation  for  the  complement  fixation  reaction  led  a  number  of 
authorities  to  the  belief  that  the  positive  Bordet-Gengou  reaction  was  in  reality  no 
amboceptor  action,  but  a  result  of  a  similar  precipitation  process.  Wassermann  and 
Bruck,  Liefmann,  Wassermann  and  Citron,  and  later  on  Moreschi  himself  realized  that 
this  physical  explanation  was  incorrect,  inasmuch  as  complement  fixation  took  place 
even  if  all  precipitation  was  prevented  by  heat  or  other 'influences.  Furthermore, 
complement  binding  of  an  unspecific  nature  can  be  produced  by  the  mixture  of  glycogen 
or  peptone  with  serum,  a  procedure  wherein  surely  no  precipitation  plays  any  part. 
Finally  Moreschi  showed  that  there  were  strongly  precipitating  sera  which  nevertheless 
did  not  exhibit  the  Bordet-Gengou  phenomenon. 

Thus  was  definitely  established  that  the  complement  fixation  was 
entirely  independent  of  either  bacteriolysis  or  precipitation. 

Following  Moreschi's  researches,  Neisser  and  Sachs  continued  Gengou's 
studies  and  advised  this  demonstration  of  the  proteid  amboceptors  as  a 
control  to  the  precipitation  method  for  the  differentiation  of  proteids.  Its 
action  is  so  much  finer,  and  more  delicate  than  the  precipitin  test  that  even 
the  minutest  traces  of  proteid  can  be  recognized. 

With  the  encouraging  results  of  Neisser  and  Sachs  in  mind,  Wassermann 
attempted  by  the  use  of  highly  immune  antibacterial  serum  to  discover 
any  soluble  bacterial  proteids  which  may  exist  in  the  blood,  derived  from 
the  respective  bacteria  invading  the  organism  at  the  onset  of  an  infection. 
Practical  application  proved  that  not  enough  of  these  proteids  existed  free 
in  the  circulation,  but  that  they  were  probably  bound  by  the  tissue  cells. 

Wassermann  and  Bruck  then  employed  the  complement  fixation  test 

with  the  idea  of  demonstrating  the  existence  of  the  respective  antigens  in 

the  diseased  organs.     Tuberculous  glands  and  lungs  served  as  material  for 

this  experiment.     They  were  able  to  obtain  complement  fixation  when  an 

extract  of  tuberculous  organs  as  antigen  was  mixed  with  a  tuberculous 

serum  (manufactured  by  the  Hochst  Farbwerke) .     If  instead  of  the  latter, 

the '  serum  from   tuberculous  individuals  was  substituted,   no  positive 

complement  fixation  reaction  was  obtained.     On  the  other  hand,  the 

reaction  was  given  if  the  human  tuberculous  serum  employed 

Antituber-    came  from  an  individual  who  had  received  therapeutic  inocu- 

culin.  lations  of  tuberculin.  In  other  words,  the  serum  of  treated 
individuals  contained,  in  contrast  to  the  untreated  ones, 
amboceptors  against  a  soluble  tuberculous  substance  also  present  in  the 
extract  of  tuberculous  glands.  Wassermann  and  Bruck  identified  this 
substance  as  tuberculin,  because  the  sera  of  the  treated  individuals  gave 
the  same  positive  results  if  a  solution  of  old  or  new  tuberculin  was  used  in- 


156  METHOD  OF  COMPLEMENT  FIXATION 

stead  of  the  extract  of  tuberculous  organs.  Thus,  the  latter  contained 
tuberculin  while  the  sera  of  the  tuberculin- treated  individuals  contained 
amboceptors  designated  by  Wassermann  and  Bruck  as  "antituberculin." 
The  name  antituberculin  has  not  been  a  well  chosen  one,  because  it  creates 
the  impression  among  many  as  being  an  antitoxin.  It  is  better  to  speak  of 
it  as  antituberculin  amboceptors. 

Since,  according  to  Wassermann  and  Bruck  these  antituberculin  ambo- 
ceptors were  not  supposed  to  be  formed  spontaneously  in  tuberculous  indi- 
viduals, but  only  in  those  treated  with  tuberculin  their  demonstration 
could  be  of  no  apparent  diagnostic  value.  On  the  other  hand,  their  exist- 
ence greatly  furthered  the  understanding  of  Koch's  tuberculin  reaction,  as 
most  tuberculous  individuals  who  had  antituberculin  amboceptors  in  their 
serum  did  not  respond  to  the  subcutaneous  injection  of  tuberculin. 

Wassermann  and  Bruck,  moreover,  showed  that  a  mixture  of  tuberculin 
with  an  extract  from  tuberculous  organs  bound  complement.  From  this 
they  concluded  that  the  extract  likewise  contains  antituberculin  ambo- 
ceptors. Thus  reasoning  they  developed  their  tuberculin  theory. 

The  difference  in  the  reaction  observed  in  a  normal  and  tuberculous 
Tuberculin  individual  after  inoculation  with  tuberculin,  can  be  fully  explained  by  the 
Theory  of    presence  of  antituberculin  amboceptors  in  the  tuberculous  focus.     By 
Wassermann  virtue  of  their  specific  affinity,  the  amboceptors  attract  the  injected 
and  Bruck.  tuberculin   toward   them.     The   tuberculin   and   antituberculin   unite, 
and  absorb  the  complement  from  the  circulating  blood  stream,  since  the 
complementophile  group  of  the  amboceptor  is  free  and  unbound.     By  virtue  of  the 
fresh  complement  which  is  an  actively  lytic  ferment,  and  the  attracted  leucocytes,  a 
partial  destruction  and  casting  off  of  the  tuberculous  focus  results.     Upon  this  depends 
the  therapeutic  effect  of  the  tuberculin.     During  a  prolonged  treatment  with  tuber- 
culin, the  body  produces  an  excess  of  antituberculin  amboceptors  so  that  finally  some 
appear  free  within  the  blood  serum.     When  this  is  the  case  the  tuberculous  organism 
loses  its  power  to  react  toward  tuberculin,  as  the  latter  is  neutralized  in  the  blood- 
stream at  a  point  away  from  the  local  focus.     No  therapeutic  effect  is  any  longer 
obtained  from  the  tuberculin  injections,  so  that  they  can,  for  a  time,  be  suspended. 
The  aim  of  tuberculin  therapy  should  be  to  work  with  small  doses  so  that  only  a  focal 
reaction  is  obtained  and  the  hyperproduction  of  antituberculin  amboceptors  be  post- 
poned as  long  as  possible. 

Numerous  exceptions  were  at  once  taken  to  this  theory  and  its  experimental  data, 
the  most  important  of  which  can  here  be  mentioned. 

"  Summier- 

d"  (E     ^6^  an(*  Nakayama  disagreed  with  the  proof  of  the  existence  of  "anti- 
tuberculin"  in  the  organ  extracts,  on  the  basis  that  Wassermann  had 

overlooked  the  effect  of  a  summation  of  antigen.     This  is  best  explained 
taken  on         .„„,.. 

as  follows:     Complement  is  bound  not  only  by  antigen  +  amboceptor, 

but  also  by  large  doses  of  antigen  itself  dependent  upon  the  normally 
.       present  amboceptors  existing  in  the  serum  employed  for  complement 

Antigen.) 


COMPLEMENT   FIXATION   DUE    TO    SUMMATION   OF   ANTIGEN 


157 


Old  tuberculin 

Complement 

Erythrocytes 

Hemolysin 

Result 

0.05 

O.  I 

0.15 

O.  I 
O.  I 
O.I 

i  c.cm.  5% 
i  c.cm.  5% 
i  c.cm.  5% 

Twice  the  hemolytic  titer. 
Twice  the  hemolytic  titer. 
Twice  the  hemolytic  titer. 

Hemolysis. 
Hemolysis. 
No  hemolysis. 

0.15  old  tuberculin  is  thus  sufficient  of  its  own  accord  to  bind  complement.     In 
their  experiment,  Wassermann  and  Bruck  found  that,  for  example, 


Tuber- 
culin 

Extract  of 
tuberculous 
organs 

Comple- 
ment 

Erythrocytes 

Hemolysin 

Result 

O.I 
O    I 

O.I 

O.I 
O    I 

i  c.cm.  5% 
i  c.cm.  5% 

Twice  the  hemolytic  dose. 
Twice  the  hemolytic  dose. 

Complement 
fixation. 
Hemolysis. 

O    I 

O    I 

i  c  cm  5% 

Twice  the  hemolytic  dose 

Hemolysis. 

This,  however,  in  no  way  proves  the  existence  of  "antituberculin"  in  the  extract  of 
tuberculous  organs,  as  it  is  perfectly  possible  and  even  probable  that  o.i  of  the  organ 
extract  contains  0.05  c.cm.  at  least  of  tubercle  bacillus  substance  (tuberculin)  which, 
when  added  to  o.i  of  tuberculin  used  for  antigen,  is  sufficient  to  give  an  amount  of 
tuberculin  perfectly  capable,  as  has  been  seen,  of  binding  complement  by  its  own 
activity. 

In  order  to  overcome  this  possibility  one  must  work  with  such  small 
but  at  the  same  time  maximum  amounts  of  antigen  and  antibodies,  that  at 
least  double  the  quantity  of  each  of  these  reagents  does  not,  of  its  own 
accord,  bind  complement.  For  tuberculin  this  is  estimated  as  follows: 


Tuberculin 

Complement 

Hemolysin 

Erythrocyte 

Result 

O.  2 

o. 

2-X  Hemolytic  dose. 

c.cm.  5% 

No  hemolysis. 

0.18 

o. 

2  X  Hemolytic  dose. 

c.cm.  5% 

No  hemolysis. 

0.15 

o. 

2  X  Hemolytic  dose 

c.cm.  5% 

No  hemolysis. 

•      d.  14 

0. 

2  X  Hemolytic  dose. 

c.cm.  5% 

Incomplete  hemolysis. 

0.  12 

0. 

2  X  Hemolytic  dose. 

c.cm.  5% 

Complete  hemolysis. 

O.  I 

o. 

2  X  Hemolytic  dose. 

c.cm.  5% 

Complete  hemolysis. 

Twelve-hundredths  is  the  maximum  non-binding  or  hemolytic  dose. 
For  the  complement  fixation  test  where  the  object  is  to  demonstrate  anti- 
tuberculin  amboceptors,  the  maximum  amount  of  antigen  to  be  used  is 
therefore  0.06  T.  or  one-half  of  the  maximum  non-binding  dose. 

In  the  same  way  the  hemolytic  dose,  and  the  dose  of  the  antibody 
should  be  estimated. 


158 


METHOD   OF   COMPLEMENT  FIXATION 


Organ 
extract 

Complement 

Hemolysin 

Erythrocyte 

Result 

0.  2 

o.  16 

O.  I 

O.I 
O.  I 
O.  I 

. 
2  X  Hemoly  tic  dose. 
!2XHemolytic  dose. 
2  X  Hemoly  tic  dose. 

i  c.cm.  5% 
i  c.cm.  5% 
i  c.cm.  5% 

o 
Complete  hemolysis. 
Complete  hemolysis. 

The  non-binding  dose  is  0.16.  The  amount,  however,  to  be  employed 
in  the  complement  fixation  test  must  be  o.c8  c.cm.  of  organ  extract. 

If  on  mixing  0.06  T.  and  0.08  extract,  complement  fixation  still  appears, 
then  this  summation  of  antigen  can  be  disregarded  and  an  antigen  antibody 
reaction  must  be  considered.  For,  even  granting  that  0.08  of  extract  does 
for  its  greater  part,  e.g.,  0.06  at  the  most,  consists  of  tuberculin,  then  this 
amount  +  0.06  of  the  tuberculin  in  the  antigen  only  makes  0.12  of  tuber- 
culin, a  quantity  not  sufficient  to  fix  the  complement.  De  facto,  comple- 
ment fixation  does  occur  when  the  above  test  is  carried  out  with  proper 
dosage,  so  that  most  probably  it  is  occasioned  by  the  biological  antigen 
antibody  reaction.  As  a  general  rule  for  all  complement  fixation  tests,  the 
dose  of  each  ingredient  employed  should  never  be  more  than  1/2  of  its  maximum 
quantity  that  does  not  of  itself  bind  complement. 

A  second  exception,  taken  by  Weil  and  Nakayama  as  well  as 
Other       by  Morgenroth  and  Rabinowitsch  relates  to  the  activity  of  the 
Exceptions,  complement   when   it   combines   with   tuberculin    and   anti- 
tuberculin. 

They  claim  that  by  this  union  the  complement's  lytic  func- 
tion is  entirely  lost.  Morgenroth  and  Rabinowitsch  even  go  so  far  as  to 
deny  the  existence  of  anti tuberculin  in  the  blood  of  tuberculous  individuals. 
The  author  also  undertook  a  minute  study  of  this  question  and  came  to 
the  following  definite  conclusion. — There  are  some  tuberculous  individuals 
who  spontaneously  develop  antituberculin  amboceptors,  a  fact  to  be  ex- 
pected because  it  has  for  a  long  time  been  known  that  on  and  off  tuberculin 
can  be  liberated  in  the  organism  of  tuberculous  individuals.  As  a  natural 
consequence  antibodies  will  be  formed,  and  most  probably  by  those  tissue 
cells  in  the  neighborhood  of  the  liberation  of  the  tuberculin,  i.e.,  the  focus 
of  infection. 

Before  proceeding,  however,  to  the  author's  conception  of  the  tuberculin 
theory  it  is  necessary  to  review  Ehrlich's  principles  of  immunity  upon  which 
the  ideas  of  antibodies  and  their  specificity  are  based. 

Ehrlich's  idea  of  the  biological  structure  of  cells  is  that  they 

Ehrlich's    consist  of  two  parts,  a  central  functionating  radicle  ("Leist- 

Side  Chain  ungskern")  upon  which  depends  the  specialized  activities  of 

Theory,     the  cells,  as  for  example,  a  glandular  or  nerve  cell,  and  a 

multiplicity  of  side  chains  or  receptors  (a  term  borrowed  from 


the  chemistry  of  the  benzol  group),  by  means  of  which  the  cell  enters  into 
chemical  relation  with  food  and  other  substances  brought  to  it  by  the  circu- 
lation. These  receptors  are  exceedingly  numerous,  as  the  nutritive  sub- 
stances upon  which  the  cell  depends  for  its  maintenance  are  very  varied. 
Besides  these  general  receptors  the  special  cells  also  have  different  and 
special  side  chains;  then,  too,  there  exist  very  great  quantitative  differ- 
ences among  the  latter;  and  finally  it  must  be  added  that  the  selective 
activity  of  the  cells  depends  upon  the  variability  of  these  receptors. 

When  an  infection  occurs,  pathological  material  is  brought  to  the  cell 
bodies  instead  of  physiological  normal  substances.  Certain  of  these  poi- 
sonous products  find  suitable  receptors  in  all  of  the  cell  groups,  others  fit 
only  into  distinct  groups  of  cells,  while  a  third  class  are  not  taken  up  at  all. 
The  organism  which  possesses  no  receptors  for  any  of  the  pathological 
agents  cannot  assimilate  any  deleterious  substances  and  is  therefore  im- 
mune. Lack  of  amboceptors  is  therefore  a  natural  form  of  immunity. 
The  organism,  having  only  a  special  group  of  cells  for  the  reception  of  cer- 
tain pathological  matter,  will  make  use  of  these  cells  for  the  binding  and 
assimilation  of  the  toxic  material.  For  example,  the  nerve  cells  alone  have 
receptors  for  tetanospasmin ;  no  matter  how  or  when  the  poison  is  intro- 
duced into  the  organism  the  nerve  cells  will  absorb  it.  As  this  toxin  is 
poisonous  for  the  central  atom  group  (Leistungskern)  of  the  nerve  cell,  the 
latter  is  destroyed.  The  union  between  the  nerve  cell  receptors  and  the 
tetanospasmin  toxin  is  only  the  preliminary  act  for  the  cell  destruction; 
the  actual  death  of  the  cell  being  caused  by  the  action  of  the  toxophore 
group  of  the  poison  upon  the  functional  radicle  of  the  cell.  If,  however, 
such  receptive  side  chains  are  possessed  not  only  by  the  brain  but  also  by 
other  cells,  e.g.,  connective-tissue  cells,  the  tetanospasmin  will  in  part  be 
bound  by  the  latter.  The  toxophore  group  of  the  toxin  does  not  have 
any  harmful  effect  upon  the  functional  radicle  of  these  cells,  and  thus  no 
toxic  effects  will  be  incurred  by  the  union,  and  the  nerve  cells  remain 
unaffected. 

The  number  of  receptors  which  cells  possess  for  tetanospasmin,  for 
example,  are  limited  and  after  their  junction  with  the  tetanospasmin,  are 
rendered  useless  and  inactive.  By  the  normal  repa.rat.ive  mechanism  of 
the  body,  new  receptors  are  generated.  This  reparative  process  does  not 
as  a  rule  stop  at  a  simple  replacement  of  lost  elements,  but  according  to 
the  hypothesis  of  Weigert  tends  to  overcompensation.  The  receptors 
eliminated  by  toxin  absorption  are  reproduced  in  an  excess  of  the  simple 
physiological  needs  of  the  cell.  Continuous  and  increasing  dosage  of  the 
toxin  soon  leads  to  such  excessive  production  of  receptors  that  the  latter 
find  no  more  room  to  be  attached  to  the  cell,  but  are  cast  off  and  circulate 
free  in  the  blood.  They  still,  however,  retain  their  property  of  being  able 
to  combine  with  tetanospasmin. 


l6o  METHOD   OF  COMPLEMENT  FIXATION 

If  such,  an  organism  is  injected  with  tetanospasmin  the  latter  toxin  is 
bound  by  the  free  receptors  in  the  serum,  and  thus  the  respective  " sessile" 
receptors  attached  to  the  cells  are  prevented  from  coming  in  contact  with 
the  poison.  Inasmuch  as  the  free  receptors  possess  no  functional  radicle 
which  can  be  injured,  the  toxin  remains  entirely  innocuous  for  the  indi- 
vidual. Such  protective  bodies  lend  to  the  organism  its  attained  immunity 
and  are  known  as  antitoxins.  Their  function  can  be  compared  to  lightning 
rods. 

v.  Behring  well  expresses  their  action  when  he  states  that  the  same 
elements,  which  attached  to  the  cells  render  the  body  susceptible  to  toxic 
substances,  when  circulating  freely  in  the  blood  serve  to  protect  it. 

The  antibodies  against  toxins  and  ferments  are  of  the  simplest  form. 
They  possess  only  a  binding  group  which  has  an  affinity  toward  the  hapto- 
phore  group  of  the  toxins  and  ferments.  They,  therefore,  belong  to  the 
class  designated  by  Ehrlich  as  "haptines"  of  the  first  order. 

To  the  haptines  of  the  second  order  belong  the  agglutinins  and  pre- 
cipitins.  They  possess  besides  a  haptophore  group  also  an  agglutinophore 
or  precipitinophore  group  by  virtue  of  which  agglutination  or  precipitation 
takes  place. 

Belonging  to  the  haptines  of  the  third  order  are  the  class  of  amboceptors 
which  have  in  addition  to  the  haptophore  group  also  a  complementophile 
group  for  their  union  with  the  complement. 

These  hypotheses  of  Ehrlich  greatly  simplify  the  explanation 
Citron's      of  many  serum  reactions  as  well  as  many  of  the  phenomena 
Tuberculin    associated  with  the  action  of  tuberculin.     In  all  probability 
Theory,      the  healthy  cells  which  exist  in  the  tuberculous  focus  and 
which  are  capable  of  reaction,  produce  the  antituberculin. 
Christian  and  Rosenblatt  offered  experimental  evidences  for  this  state- 
ment.    They  demonstrated  that  tuberculous  guinea-pigs,  in  whom  anti- 
tuberculin  was  produced  by  tuberculin  injections,  showed  a  diminution 
of  antituberculin  in  the  blood  when  tuberculous  glands  were  removed  by 
operation. 

The  antituberculin  production  by  the  cells  is  a  transitory  action  arising 
only  when  tuberculin  has  spontaneously  or  artificially  reached  the  circu- 
lation. Following  this  stage  of  activity  there  comes  a  period  of  quiescence 
during  which  no  free  antituberculin  can  be  found  in  the  serum.  The  cells, 
however,  are  supplied  with  a  great  many  more  sessile  receptors  than  usually: 
they  possess  a  higher  affinity  toward  tuberculin  and  produce  antituberculin 
much  more  readily  than  normal  cells. 

This  also  explains  why  the  smallest  amounts  of  tuberculin  produce  a 
reaction  in  tuberculous  and  not  in  the  normal  individuals.  In  the  former, 
the  cells  in  the  zone  surrounding  the  tuberculous  focus  are  abundantly 
supplied  with  receptors,  so  that  on  the  injection  of  tuberculin,  its  action 


CITRON'S  THEORY  FOR  THE  TUBERCULIN  REACTION  161 

appears  almost  concentrated  at  this  point.  Occasionally  the  sessile  recep- 
tors are*  relatively  scarce  and  the  first  injection  excites  no  reaction.  By 
the  time  of  the  second  or  third  inoculation  these  sessile  amboceptors  have 
so  increased  that  a  positive  reaction  is  apparent  when  the  same  or  even  a 
smaller  dose  is  injected.  This  phenomenon  of  increased  sessile  receptors 
explains  the  reappearance  of  subsided  subcutaneous,  cutaneous,  or  oph- 
thalmo  reactions  after  renewed  injections  of  tuberculin. 

To  recapitulate  the  biological  phenomena  associated  with  a  positive 
tuberculin  reaction,  it  may  be  said  that  the  tubercle  bacilli,  or  portions  of 
their  body  substances  existing  in  the  infected  focus,  stimulate  the  adjacent 
cells  to  produce  a  great  number  of  sessile  receptors.  When  the  tuberculin 
is  injected  for  the  first  time,  these  sessile  receptors  at  once  take  up  the 
tuberculin  and  as  a  result,  the  production  of  an ti tuberculin  in  the  focus  is 
further  stimulated. 

A  part  of  the  tuberculin  has  already  been  attracted  by  the  receptors  of 
the  cutis  or  subcutis  cells  (intracutaneous  reaction)  or  the  cells  of  the  mu- 
cous membrane  (ophthalmo  reaction)  and  here  too  has  stimulated  the 
production  of  antibodies  (an  ti  tuber  culm).  It  is  only  a  quantitative  differ- 
ence in  the  number  of  receptors  which  actually  differentiates  a  normal  from 
a  tuberculous  individual.  Thus  is  explained  that  even  in  non-tuberculous 
individuals  a  local  reaction  may  be  obtained  if  the  dose  of  tuberculin  in- 
jected is  large  enough;  a  focal  reaction,  however,  will  be  given  only  by  a 
tuberculous  subject.  The  greater  the  number  of  sessile  antituberculin 
receptors  that  have  been  formed  in  the  tuberculous  focus,  the  greater 
becomes  the  affinity  of  these  cells  toward  the  tuberculin;  so  that  with  the 
second,  third,  and  subsequent  tuberculin  injections,  focal  reactions  (i.e., 
antituberculin  productions)  are  more  easily  stimulated. 

As  for  the  origin  of  the  fever,  it  is  probable  that  a  pyrotoxic  substance 
is  formed  by  the  union  between  tuberculin,  antituberculin  and  comple- 
ment. This  poison  first  isolated  in  vitro  by  Citron  will  again  be  referred 
to  and  belongs  to  the  class  of  anaphylotoxins  (Friedberger)  or  toxopeptids 
(M.  Wassermann  and  Keyser). 

Finally  the  antituberculin  receptors  become  so  numerous  that  they 
are  detached  from  the  cells  and  become  free  receptors.  This  period,  how- 
ever, is  only  transitory,  as  is  corroborated  by  the  difficulty  connected  with 
the  demonstration  of  these  antibodies  in  the  focus.  This  free  antituber- 
culin combines  with  the  tuberculin  (spontaneously  formed  or  injected) 
and  attracts  the  complement,  or  the  complement  producing  phagocytes. 
Uncombined  complement  has  no  effect  on  the  tissues.  It  is  different,  how- 
ever, with  the  phagocytes.  These  can  without  any  additional  help  act 
directly  upon  the  infected  focus.  If  the  tuberculin  treatment  is  continued, 

*  A  sessile  receptor  is  one  which  is  still  attached  to  its  cell  and  not  yet  free  in  the  blood. 


1 62  METHOD   OF   COMPLEMENT  FIXATION 

a  period  arises  during  which  the  antituberculin  bodies  are  so  greatly  ac- 
cumulated in  the  local  focus  that  they  ultimately  escape  into  the  blood 
stream.  This  freely  circulating  antituberculin  neutralizes  any  freshly 
injected  tuberculin,  so  that  such  patients  become  refractory  against  even 
the  largest  amounts  of  it.  (Tuberculin  immunity.)  Tuberculin  immunity 
is  not,  however,  in  all  cases  to  be  identified  with  a  strong  antituberculin 
content  in  the  serum  as  is  demonstrated  by  the  complement  fixation 
method.  For  example,  it  is  very  difficult  to  produce  antituberculin  bodies 
by  treatment  with  S.  B.  E.,  although  by  its  use  an  immunity  against  B.  E. 
is  easily  attained. 

The  question  as  to  how  great  a  role  the  antituberculin  bodies  play, 
and  their  exact  interpretation  is  very  complicated.  The  present  status 
of  our  knowledge  may  be  expressed  as  follows. 

The  administration  of  the  various  tuberculin  preparations  to  tubercu- 
lous patients  results  in  the  formation  of  antibodies  within  their  serum, 
which  with  the  respective  tuberculin  as  antigen  will  give  the  phenomenon 
of  complement  fixation  in  vitro.  The  different  tuberculins  are  not  equally 
efficient  as  antigens.  Thus  B.  E.  is  the  best  stimulant  of  antibodies. 
Furthermore  the  antibodies  obtained  from  the  soluble  tuberculin  prepa- 
rations are  not  identical  with  those  from  the  insoluble  products.  Some 
sera  fix  complement  only  with  old  tuberculin,  others  react  only  with  new 
tuberculin.  The  tuberculous  serum  of  Meyer  and  Ruppel  contains  be- 
sides these  two  antibodies  another  group  which  gives  the  complement 
fixation  test  with  an  alcoholic  extract  of  tubercle  bacilli  as  antigen  (Citron 
and  Klinkert).  It  may  be  assumed  that  with  other  antigens,  other  com- 
plement binding  antibodies  will  be  discovered.  The  presence  of  these 
antibodies  is  of  itself  no  criterion  for  the  existence  of  a  tuberculin 
immunity.  There  are  tuberculous  subjects  who  are  not  susceptible  to 
tuberculin  and  at  the  same  time  possess  no  in  vitro  demonstrable  anti- 
bodies; reversely,  there  are  very  highly  susceptible  tuberculous  individuals 
with  antibodies  in  their  serum.  The  explanation  for  the  last  class  of  cases 
has  been  furnished  by  Citron.  The  author  demonstrated  that  the  anti- 
tuberculin  contained  within  the  serum  in  certain  instances  raises  the  sus- 
ceptibility against  tuberculin.  Thus  there  is  a  hypersusceptibility  of  a 
humoral  form  analogous  to  serum  anaphylaxis,  besides  the  hypersensi- 
tiveness  depending  upon  the  increased  number  of  sessile  receptors. 

To  offer  an  explanation  for  the  first  class  of  cases  (i.e.,  tuberculous  sub- 
jects with  no  antituberculin  and  not  susceptible  to  tuberculin)  it  has  been 
assumed  that  besides  complement  binding  agents  there  are  also  directly 
neutralizing  or  antitoxin-like  bodies  within  the  serum.  Pickert  and  Low- 
enstein  could  demonstrate  that  the  serum  of  some  tuberculous  patients 
had  the  property  when  mixed  with  tuberculin  to  so  neutralize  the  latter 


i63 

that  it  could  no  longer  be  used  for  the  cutaneous  reaction.     They  named 
this  antibody  "antikutine,"  and  proved  its  existence  as  follows: 

Twenty  days  after  the  last  tuberculin  injection  the  serum  of  the  patient 
is  withdrawn  and  mixed  with  tuberculin  in  i,  2,  5  and  10  per  cent,  tuber- 
culin serum  mixtures.  These  are  kept  in  the  incubator  for  two  hours,  then 
in  the  ice-box  for  twenty  hours  and  then  used  for  the  cutaneous  reaction. 
The  abrasions  made  with  the  v.  Pirquet  borer  must  be  at  least  3  cm.  dis- 
tant from  each  other,  intervened  by  control  spots.  By  gently  stretching 
the  skin  of  the  forearm  one  can  apply  ten  reactions  over  this  area.  Only  a 
distinct  papule  formation  should  count  as  a  positive  reaction  and  ob- 
servation should  be  continued  for  six  days.  Just  as  antituberculin 
amboceptors  may  be  found  in  the  serum  of  patients  who  have  not  had 
any  specific  treatment,  so  also  can  these  antikutine  bodies  originate 
spontaneously. 

Marmorek  described  still  another  antibody,  found  in  a  horse  immunized 
with  living  young  tubercle  bacilli.  This  serum  when  mixed  with  the  urine 
from  patients  with  active  febrile  tuberculosis  will  fix  complement.  Exam- 
inations by  Citron  and  Klinkert  showed  that  here  one  is  probably  dealing 
with  an  antibody  brought  about  by  immunization  against  altered  tuber- 
culous tissue. 

In  former  times  a  negative  tuberculin  reaction  after  a  prolonged  treat- 
ment was  stamped  as  a  cure  of  the  tuberculosis,  a  fact  obviously  incorrect; 
for  no  matter  how  successful  the  tuberculin  therapy  may  be,  it  cannot 
always  be  considered  as  a  complete  curative  procedure.  In  general,  that 
method  should  be  adopted  which  makes  the  individual  non-susceptible  to 
the  largest  doses  of  tuberculin.  It  was  found  in  practice  that  those  patients 
having  the  greatest  amount  of  antituberculin  in  their  serum  usually 
offered  a  better  prognosis. 

The  experiences  gained  by  the  employment  of  the  complement 

Serum       fixation  test  in  tuberculosis  lead  to  its  application  in  the  study 

Diagnosis  of   of  syphilis.     The  difficulties  in  this  disease  were  greater,  inas- 

Syphilis.     much  as  there  were  no  bacteria  or  preparations  like  tuberculin 

which  could  be  used  as  antigen. 


Syphilitic  human  organ  extracts  were  employed,  with  the  idea  that  these  would 
contain  the  specific  virus.  The  serum  of  monkeys  previously  immunized  with  such 
extracts,  when  mixed  in  vitro  with  the  latter,  gave  complement  fixation.  '  This  experi- 
ment is  not,  however,  conclusive;  the  positive  reaction  may  be  due  to  anti-human 
proteid  amboceptors  produced  at  the  same  time  by  the  injection  of  the  human  serum 
contained  in  the  organ  extract.  The  experiment  was  changed  and  the  syphilitic  organ 
extracts  from  apes  were  used  so  as  to  exclude  this  error.  Even  in  this  way  complement 
fixation  was  attained.  Later  on  it  was  found  unnecessary  to  inject  the  monkeys  with 
the  extracts  since  after  ordinary  infection  their  serum  would  give  complement  fixation. 
In  this  manner  it  was  almost  definitely  established  firstly,  that  these  extracts  contained 


164  METHOD   OF   COMPLEMENT  FIXATION 

a  substance  specific  for  syphilis  which  could  with  most  probability  be  considered  a 
luetic  antigen,  and  secondly  that  infected  apes  possess  antibodies  against  this 
antigen. 

The  next  step  was  to  try  the  reaction  in  man.  The  first  experiments  of 
Wassermann,  Neisser,  Brack  and  Schucht  did  not  give  the  hoped-for 
returns.  Although  the  reaction  was  obtained  with  human  serum,  the 
percentage  of  positive  results  was  so  small  (see  next  chart)  that  its  practical 
value  as  a  means  of  diagnosis  offered  no  great  help.  Only  in  general 
paralysis  did  the  expectation  seem  promising.  In  about  80  per  cent,  of  all 
cases  Wassermann  and  Plaut  were  able  to  demonstrate  the  luetic  anti- 
bodies in  the  cerebrospinal  fluid. 

Schiitze's  experiments  in  tabes  led  him  to  the  same  findings.  Citron  has  obtained 
a  much  smaller  percentage  of  positive  reacting  cerebrospinal  fluids  in  tabes. 

As  it  seemed  that  the  means  of  diagnosis  was  not  to  be  established  by 
the  demonstration  of  the  syphilitic  antibody,  Neisser  and  Bruck  believed 
that  better  results  may  possibly  be  achieved  by  the  discovery  of  the  luetic 
antigen  in  the  serum  through  complement  fixation. 

This  attempt  too  was  unsuccessful.  No  antigen  could  be  found,  but  the  extracts 
of  red  blood  cells  from  syphilitic  individuals  when  mixed  with  the  serum  of  highly 
immunized  monkeys  gave  a  positive  complement  fixation.  Neisser  and  his  co-workers 
concluded  therefrom  that  the  erythrocyte  extract  contained  the  luetic  antigen. 
Citron  soon  demonstrated  that  the  extracts  of  normal  individuals  gave  a  similar  reac- 
tion and  what  was  more  important,  that  this  so-called  blood  antigen  existed  in  the 
blood  entirely  uninfluenced  by  mercurial  treatment.  Since  these  experiments,  not 
much  importance  has  been  attached  to  this  reaction. 

Meanwhile  the  author  working  at  the  Kraus  clinic  proved  by  a  large 
series  of  experiments  that  luetic  antibodies  were  present  in  almost  all  cases 
of  lues.  The  reaction  is  dependent  upon  two  rules. 

The  First. — The  longer  the  syphilis  virus  has  acted  upon  the  organism 
and  the  more  numerous  its  recurrent  manifestations  have  been,  the  more 
frequently  will  a  positive  reaction  be  obtained  and  the  stronger  will  the 
antibody  content  of  the  serum  be. 

The  Second. — The  sooner  a  proper  mercury  therapy  is  instituted,  the 
more  often  it  is  repeated,  and  the  shorter  the  interval  since  the  last  treat- 
ment, the  smaller  will  the  antibody  content  of  the  serum  be  and  the  greater 
the  possibility  of  a  negative  reaction. 

These  points  were  soon  corroborated  by  numerous  other  workers  in  the 
field,  so  that  at  the  present  day  they  can  be  taken  as  absolute  facts.  The 
following  chart  will  explain  some  of  the  statements  aforementioned. 


WASSERMANN   REACTION   IN   LUETIC   INFECTIONS 


First  period 


Second  period 


*,£ 

'Si   ^ 

T3 

"-J3 

T3 

Citron 

d 

3 

£   3 

q 

^9 

e 

M 

8 

z  £ 

ce 

c3 

rrt 

CJ 

<-! 

£ 

Wassermann,  ! 
ser,  Bruck  &  Scl 

Wassermann  ; 
Plaut 

J 

T3 

§ 

4 

G3 
^ 

Morgenroth  ; 
Stertz 

Schiitze 

I 

I 
% 

<N 

.  ^    r^ 
MO 
|J 

C/2 

1 

c« 

1 
M 

Fleischmam 

1 

Ledermann 

o 

1 

td 

o 

Lues  I 

8 

% 

% 

% 

% 

% 

% 

oo 

48.  2 

IOO 

68 

<2    6 

% 

Lues  II 

26.7 

' 

y^ 
08 

7O 

Q-2 

Q2 

o*  •  w 

ICO 

Q~»   8 

with 

y<j 

/  y 

yo 

yj 

y^  •  ** 

symp- 

toms 

.without 

14.6 

80 

20 

6/L 

7=;  6 

4.6 

symp- 

w^. 

/  J  •  " 

•ty 

toms 

("early 

latent") 

Lues  III 

}77   ? 

'  74 

with 

21.6 

/  /  •  o 

/T- 

OI 

<7    A 

08 

IOO 

02    6 

88.9 

symp- 

y •*• 

J  /    •  T- 

yu 

y  ^  •  *•* 

toms 

without 

ii  •  3 

"?7 

2O    2 

A  2 

36.8 

77    tr 

symp- 

O 

o  / 

^r* 

o  /  •  0 

toms 

("Itae 

latent") 

j  .-;.- 

Pro- 

(80) 

(75) 

(100) 

IOO 

1 

gressive 

(spinal 

(spinal 

(spinal 

paraly- 

fluid) 

fluid) 

fluid) 

;  .•  _    *•      .  - 

I  88 

sis 

Tabes 

(66) 

^6.6 

7O 

60 

dorsalis 

y  \j\jj 

(spinal 

(22) 

/  V 

fluid) 

'spinal 

-.\'.:  •' 

fluid) 

As  has  been  repeatedly  remarked,  specificity  is  the  important  element  in 
every  biological  reaction.  The  reaction,  known  after  the  discoverer  as  the 
Wassermann  Reaction,  can  also  be  performed  if,  instead  of  the  extract  from 
luetic  organs,  an  alcoholic  extract  of  certain  normal  organs  or  certain  lipoid 
substances  is  substituted  as  antigen.  Seligmann  was  likewise  able  to  obtain 
complement  fixation  by  pure  chemical  reactions.  Consequently,  numer- 
ous authorities  expressed  the  opinion  that  the  Wassermann  Test  was  non- 
specific and  that  it  does  not  at  all  represent  an  antigen  antibody  interaction. 


i66 


METHOD   OF   COMPLEMENT  FIXATION 


There  is  no  doubt,  however,  that  this  exceptional  view  is  incorrect.  It  is  true  that 
the  real  syphilitic  antigen  is  unknown,  but  most  probably  it  is  neither  the  pure  spiro- 
chaetes  nor  a  pure  lipoid  substance.  The  author  has  expressed  the  hypothesis  that  the 
antibody  producing  antigen  is  a  toxolipoid.  This  explains  the  fact  that  pure  lipoids 
cannot  stimulate  any  antibodies,  but  can  react  nevertheless  with  luetic  antibodies  in 
vitro. 

The  accompanying  diagram  (Fig.  17)  explains  this  hypothesis.  In 
order  to  answer  the  objections  raised  against  such  a  theory,  the  author  has 
proposed  the  indifferent  term  of  "Lueseargine"  for  the  luetic  antibodies,  as 
long  as  their  biological  structure  is  unknown. 

Experiments  by  Citron  and  Munk 
prove    without    any    doubt   that    the 

Lues-Antigen  {  ^  luesreagine  is  a  true  antibody  of  an  an- 

tigen contained  only  within  the  aqueous 
syphilitic  extract.  Aqueous  and  alco- 
holic extracts  of  normal  organs  do  not 
contain  this  antigen.  Rabbits  were  im-' 
munized  with  various  antigens.  Only 
those  injected  with  watery  syphilitic 
extracts  developed  antibodies  similar 
to  the  luesreagines  of  human  syphilis 
in  that  they  reacted  with  alcoholic 
normal  extracts.  Blumenthal  and 
Meyer  corroborated  these  findings  and  further  showed  that  even  the  alco- 
holic syphilitic  extracts  do  not  contain  this  antigen. 

Since  the  cultivation  of  the  spirochetes  in  pure  culture  has  been 
simplified  by  Noguchi,  extracts  of  such  cultures  have  been  made  and 
employed  as  antigens  for  the  Wassermann  reaction.  It  was  hoped  that 
if  such  a  specific  and  efficient  antigen  could  be  obtained,  the  basis  of 
a^true  antigen  antibody  reaction  would  be  more  certain.  While  fixation 
occurs  with  the  pallida  culture  antigen,  the  results  cannot  be  depended 
upon  for  clinical  purposes;  some  cases  of  undoubted  syphilis  giving  a 
strongly  positive  reaction  with  the  syphilitic  liver  antigen,  give  abso- 
lutely negative  results  with  the  culture  antigen  (Craig  and  Nichols). 

Independent  of  the  question  of  "  biological  specificity,"  the  Wasser- 
mann reaction  must  also  be  considered  in  the  light  of  "  clinical  specificity." 
From  this  standpoint  it  fulfills  its  demands.  With  only  few  exceptions,  it 
can  be  regarded  as  absolutely  specific  for  lues. 

The  well  established  exceptions  are,  frambcesis,  trypanosomiasis,  leprosy,  malaria, 
scarlet  fever,  febris  recurrens.  The  reactions  obtained  here  are  similar,  but  not  the 
same  as  those  obtained  in  syphilis.  In  leprosy  the  difference  is  that  the  reac- 
tion can  also  be  performed  with  tuberculin  as  antigen;  in  scarlet  fever  the  reaction 
appears  only  in  a  small  percentage  of  cases  and  not  with  all  luetic  extracts.  Further- 


1 

t 

Syphilis  virus 
Lipoid  (lecithin) 
Lipoidophile  group 

A 

Amboceptor 

Complementophile 
group 

y\/W    Complement 

FIG.  17. 

SIGNIFICANCE    OF   A   POSITIVE    WASSERMANN   REACTION  167 

more,  it  disappears  at  the  latest  three  months  after  the  infection,  usually  much 
sooner.  As  for  trypanosomiasis  and  malaria  convincing  data  are  still  too  few.  In 
malaria  the  reaction  is  always  negative  if  the  parasites  are  absent. 

These  diseases  excluded,  a  positive  Wassermann  reaction  can  be  taken 
as  certain  proof  of  the  existence  of  lues.  Whether  such  a  test  is  indicative 
of  a  by-gone  infection  or  whether  it  means  that  an  active  process  is  still 
going  on,  has  been  for  a  long  time  a  subject  of  discussion.  The  author  is 
of  the  firm  opinion  that  the  demonstration  of  the  "lues  reagine"  means  active 
lues.  The  reason^  for  this  belief  are  as  follows : 

1.  The  almost  constant  presence  of  the  reaction  in  all  cases  of  manifest 
lues  excepting  primary  lesions.     During  this  initial  stage  it  is  entirely 
absent  or  only  faintly  positive.     It  appears,  however,  later  on. 

2.  The  practically  assured  existence  of  the  reaction  with  a  recurrence 
of  symptoms  even  though  formerly  it  was  negative. 

3.  The  possibility  of  influencing  a  positive  reaction  so  that  it  becomes 
negative,  by  the  use  of  mercury.     The  latter  holds  true  also  for  those  cases 
which  show  no  symptoms  and  are  therefore  incorrectly  designated  as 
latent  syphilis.     It  has  been  proven  that  such  are  in  reality  by  no  means 
latent,  but  have  an  active  process  at  some  point  escaping  detection,  as  the 
aorta.     Only  cases  of  a  nature  which  have  no  symptoms  and  a  negative 
reaction  should  be  considered  as  latent  syphilis;  those,  however,  with  no 
symptoms,  but  a  positive  reaction  as  belonging  to  the  class  of  active  lues. 

4.  The  evidence    that  apparently  healthy  individuals,  but  with  ra 
positive   reaction,   have   infected   others,  or   have   suddenly   developed 
tertiary    or   postluetic  manifestations — tabes,  paresis,  diseases    of    the 
aorta,  etc. 

An  objection  has  frequently  been  raised,  that  in  spite  of  existing  disease,  the  reac- 
tion has  been  found  negative.  If  the  statistics  covering  the  largest  number  of  cases  are 
studied,  it  will  be  seen  that  such  instances  are  rare.  Exceptions  are  discovered 
in  every  biological  reaction,  especially  one  which  is  complicated,  and  where  five  different 
agents  come  into  play;  even  in  the  immunization  of  animals  differences  will  be 
found  in  that  some  produce  a  highly  agglutinating  or  precipitating,  etc.,  serum,  while 
others  will  show  few  or  even  no  antibodies.  Individual  differences  are  prevalent  to 
such  an  extent  that  exceptions  to  the  rule  must  be  taken  for  granted.  Fortunately,  a 
negative  reaction  in  existing  lues  is  so  rare,  that  for  practical  purposes  its  possibility 
may  be  overlooked,  at  least,  with  reservation. 

As  a  general  rule,  antibodies  persist  in  an  organism  for  a  certain  time  after  infection, 
when  the  individual  has  become  perfectly  well.  Discussion,  to  the  effect  that  it  may 
be  possible  for  a  positive  Wassermann  reaction  to  similarly  signify  a  past  infection  or  a 
state  of  immunity,  has  been  raised.  But  it  must  be  said  that  immunity  in  syphilis 
is  a  condition  thus  far  unproven,  and  almost  unknown.  All  symptoms  previously 
attributed  to  such  an  immunity  can  more  easily  be  explained  in  the  light  of  a  continua- 
tion of  the  disease.  As  for  the  "lues  reagine"  remaining  after  the  cure  of  the  infec- 
tion, this  phenomenon  is  undoubtedly  possible.  The  analogy  with  other  diseases  seems 
lost,  however,  when  one  considers  that  the  syphilitic  reaction  is  discovered  thirty  or 


1 68  METHOD   OF   COMPLEMENT  FIXATION 

forty  years  after  an  infection,  while  antibodies  in  general  persist  for  weeks,  months 
or  at  the  most  for  several  years,  following  an  infection.  Still  it  may  be  possible  that 
the  syphilis  "reagine"  is  characterized  by  the  difficulty  with  which  it  is  excreted  and 
by  the  tendency  of  the  cells  when  once  stimulated  to  produce  antibodies  to  continue 
to  do  so.  The  influence  of  mercury,  however,  demonstrates  that  this  phenomenon  is 
closely  allied  to  similar  actions  exhibited  by  the  class  of  bacteria.  If  a  patient  whose 
serum  gives  a  positive  reaction  is  subjected  to  mercurial  treatment,  the  reaction  be- 
comes negative.  The  mercury  has  destroyed  the  stimulant  or  irritant  which  has  led 
the  cells  to  the  production  of  antibodies.  If  this  stimulant  is  excluded,  the  "lues 
reagine"  disappears  from  the  blood  just  as  bacterial  antibodies  disappear  after  the 
bacteria  have  been  eradicated.  Thus  there  is  no  basis  for  attributing  to  the  luetic 
antibodies  any  exceptional  properties. 

The  fact,  that  mercury  leads  to  an  alteration  in  the  reaction, 

Citron's     prompted  the  author  to  employ  the  Wassermann  test  as  a 

"Biological  guide  to  the  biological  mercurial  treatment.     The  aim  was  not 

Mercurial   only  to  cause  a  disappearance  of  all  manifestations,  but  to  obtain 

Treatment."  a  negative  reaction.     It  soon  appeared  that  a  negative  reaction 

once  obtained  did  not  necessarily  remain  so.     As  soon  as  a 

recurrence  set  in  the  reaction  became  positive  again;  in  fact,  the  reaction 

also  reappeared  without  a  return  of  symptoms.     In  the  latter  case  such  a 

return  alone  was  regarded  as  a  fresh  manifestation  of  a  reactivation  process 

and  an  indication  for  treatment.    It  became  advisable,  therefore,  to  repeat 

the  test  at  definite  intervals  and  depend  upon  the  return  of  the  reaction  for 

further  treatment.     This  basis  of  therapy,  which  at  first  met  with  marked 

opposition,  has  recently  won  many  followers. 

The  experiments  of  Boas  in  Copenhagen  are  especially  instructive 
from  this  point  of  view. 

He  examined  eighty-two  patients  with  secondary  syphilis  before  and  after  mercurial 
therapy.  All  gave  positive  reactions  before  the  treatment;  after  it,  seventy-six  gave 
no  reaction,  six  retained  the  positive  reactions;  one  of  the  six  did  not  return  for  observa- 
tion. Of  the  remaining  five,  all  had  a  return  of  symptoms  within  one  month  after 
cessation  of  the  mercury,  while  of  the  seventy-six  only  three  returned  with  a  recurrence. 
Boas  next  made  observations  of  sixty-five  patients  who  were  in  the  first  three  years  of 
their  infection,  but  who  gave  a  negative  Wassermann  after  the  treatment.  In  sixty- two 
cases,  a  positive  reaction  reappeared  after  one  to  two  months,  eight  of  these  having  at 
the  same  time  a  recurrence  of  symptoms;  of  the  remaining  fifty-four,  nineteen  were  not 
treated.  They  all  showed  a  return  of  symptoms,  but  only  one  and  a  half  months  after 
the  appearance  of  the  positive  Wassermann.  Thus  if  the  scheme  of  the  chronic  inter- 
mittent mercurial  therapy  of  Neisser  and  Fournier  were  followed,  these  patients  would 
begin  to  get  treatment  one  and  a  half  months  after  the  active  lues  had  again  started,  as 
shown  by  the  positive  Wassermann  reaction.  Of  the  remaining  thirty-five  cases  all 
began  treatment  when  the  Wassermann  test  became  positive.  None  of  these  had  any 
return  of  symptoms  during  the  following  period  of  observation  (three  to  five  months). 

The  experiments  of  Boas  show  distinctly  the  advantages  of  the  mer- 
curial therapy  when  based  upon  the  biological  reaction  instead  of  upon  the 


LUES   ASYMPTOMATICA  1 69 

schematic,  symptomatic,  chronic,  intermittent  treatment  of  Fournier  and 
Neisser. 

At  the  present  day,  when  the  spirochaetes  can  be  so  readily  found  in  the 
primary  lesion  of  syphilis,  the  biological  mercurial  treatment  should  be 
undertaken  in  the  earliest  stage.  It  is  possible  even  to  begin  at  a  time 
when  the  serum  reaction  is  still  negative,  but  after  the  spirochaetes  have 
been  demonstrated.  The  most  ideal  cases  are  those  in  which  treatment  is 
instituted  so  early  that  they  never  develop  a  positive  Wassermann  test. 

Naturally  the  statement  made  that  mercurial  treatment  should  be  continued  until 
the  reaction  becomes  negative  may  be  limited  by  certain  centra-indications  which  may 
arise  in  the  general  condition  of  the  patient.  This  must  always  be  considered.  Especial 
difficulty  to  attain  a  negative  reaction  is  encountered  in  those  cases  in  which  lues  has 
persisted  for  many  years. 

It  must  be  kept  in  mind  that  the  luetic  infection  does  not  always  present 
the  typical  clinical  picture  ascribed  to  it  in  the  text-books.  The  "Lues 
asymptomatica,"  that  is,  the  lues  apparently  presenting  no  symptoms,  is 
by  no  means  rare.  To-day  one  must  not  wait  until  the  syphilitic  patient 
comes  to  the  physician,  but  it  is  the  duty  of  the  latter  to  look  for  the  evidence  of 
syphilis  among  those  related  to  or  associated  with  infected  persons.  If  one 
proceeds  in  such  a  systematic  method  it  will  be  found  that  the  mothers  of 
syphilitic  children,  so  frequently  regarded  as  immune,  are  in  reality  not  so. 
In  such  cases,  without  any  clinical  evidence  of  syphilis,  the  Wassermann 
reaction  is  positive  in  about  56  to  75  per  cent. 

This  question  becomes  of  utmost  importance  in  the  prevention  of  lues. 
For  example  the  obligatory  examination  of  the  serum  of  wet  nurses  has 
shown  that  of  all  such  applicants  at  the  Dresden  Infant  Asylum  10  per  cent, 
gave  a  positive  reaction  (Rietschels) .  On  further  study  it  was  ascertained 
that  75  per  cent,  of  the  children  of  these  apparently  healthy  women  gave 
luetic  manifestations  immediately  or  shortly  after  birth. 

The  Wassermann  Test  also  offers  a  certain  guide  as  to  the  prognosis 
of  a  case.  Thus  the  outlook  is  unfavorable  if  in  spite  of  energetic  treat- 
ment a  reaction  remains  constantly  positive.  Even  with  the  absence  of 
all  external  lesions  such  a  condition  can  be  classified  as  a  "lues  maligna." 
As  a  rule  this  term  is  applied  to  external  obvious  syphilitic  manifestations 
that  remain  entirely  uninfluenced  by  mercurial  therapy.  When  it  is  con- 
sidered that  all  general  paralysis  patients  react  very  strongly  positive,  the 
importance  of  bringing  about  a  negative  reaction  is  sufficiently  impressed. 
Salvarsan  is  of  great  help  toward  this  aim. 

A  rapid  disappearance  of  a  positive  Wassermann  reaction  after  specific 
therapy  offers  a  good  prognosis.  This  is  the  more  favorable  the  longer 
the  negative  test  persists.  Its  continuation  over  several  years  with  no 
clinical  manifestations  can  be  accepted  as  a  cure  of  the  syphilitic  infection. 


170  METHOD   OF   COMPLEMENT  FIXATION 

Serum  Diag-  ^n  c^ose  association  with  the  serum  diagnosis  of  syphilis,  com- 

nosis  of  Dis-  plement  fixation  has  been  employed  as  a  means  for  the  diagno- 

eases  Caused  sis  of  conditions  caused  by  the  animal  parasites  and  especially 

by  Animal  by   the   echinococcus.     In   the   serum   of  patients   suffering 

Parasites.    from  faGSG  infections,  substances  are  found  closely  allied  to 

the  "lues  reagine."     They  bind  complement  with  an  antigen  consisting  of 

an  extract  of  the  respective  worms  or  hydatid  fluid. 

Ghedini,  Weinberg  and  Parvu  and  others  have  found  that  in  most  cases  of  echino- 
coccus disease,  the  reaction  is  positive.  If  by  operation  the  cyst  is  only  incised,  the  re- 
action becomes  stronger  or  in  few  cases  appears  positive  for  the  first  time.  After  com- 
plete excision  of  the  cyst,  the  reaction  disappears.  According  to  Parvu  and  Laubry,  a 
positive  test  is  found  in  the  spinal  fluid  only  when  the  echinococcus  cysts  have  invaded 
the  brain. 

Ghedini  described  similar  findings,  caused  by  the  ascaris,  ankylostoma, 
etc. 


CHAPTER  XIV. 

THE  TECHNIQUE  or  COMPLEMENT  FIXATION. 

Original  method  of  Bordet-Gengou.  Wassermann-Bruck's  modification.  Technique 
of  serum  diagnosis  for  syphilis.  Echinococcus  disease.  Serum  diagnosis  of  other  dis- 
•eases,  epidemic  meningitis,  tuberculosis,  gonococcus  infections,  typhoid  fever.  Differen- 
tiation of  proteids  according  to  Neisser -Sachs. 

I.  The  Original  Method  of  Bordet-Gengou. 

a.  The  antigen  consists  of  bacteria  grown  upon  agar  for  twenty-four 
hours  and  then  suspended  in  physiological  salt  solution  to  make  a  rather 
concentrated  emulsion. 

For  typhoid  bacteria  Bordet  and  Gengou  take  5  c.cm.  of  salt  solution  to  each  cul- 
ture of  bacteria. 

For  tubercle  bacilli  80  mg.  of  the  bacteria  are  suspended  in  i  c.cm.  of  salt  solution. 

b.  The  serum  containing  the  antibody  is  heated  for  one-half  hour  at 
56°  C.  to  destroy  the  complement. 

c.  As  complement,  the  fresh  serum  of  a  normal  animal  or  human  being 
is  used.  -r 

d.  The  hemolysin  consists  of  the  inactivated  serum  of  a  rabbit  that 
has  been  immunized  against  sheep's  or  goat's  erythrocytes,  or  the  serum 
of  a  guinea-pig  injected  with  rabbit's  red  blood  cells. 

e.  The  respective  red  blood  corpuscles  are  washed,  to  free  them  of 
their  complement  containing  serum. 

A  definite  amount  of  bacterial  suspension  is  mixed  with  varying  amounts  of  in- 
activated immune  serum  and  a  proportional  amount  of  complement  is  added.  These 
three  ingredients  are  mixed  and  allowed  to  remain  at  room  temperature  for  four  to 
five  hours.  During  this  time  the  complement  is  fixed  if  the  antigen  and  antibody  are 
of  a  homologous  nature.  In  order  to  see  whether  this  union  has  taken  place  or  not, 
hemolysin  and  erythrocytes  are  added  in  a  mixture  thus  prepared:  2  c.cm.  of  inactivated 
hemolysin  +  twenty  drops  of  washed  blood  cells  are  mixed  and  allowed  to  remain  to- 
gether for  about  fifteen  minutes  so  that  the  erythrocytes  are  sensitized,  i.e.,  united  with 
the  hemolytic  amboceptor.  Of  this  mixture  each  tube  receives  o.i  to  0.2  c.cm.  If 
the  complement  has  not  become  fixed,  hemolysis  occurs  in  several  minutes.  If  the 
complement  has  become  fixed,  hemolysis  does  not  occur;  since,  however,  the  hemolysin 
also  contains  hemagglutinin,  the  erythrocytes  are  agglutinated  and  sink  to  the  bottom 
of  the  tubes; 

As  control  tests,  Bordet  and  Gengou  considered  the  following  very  necessary: 
i.  Bacterial  suspension  +  inactivated  normal  serum  (instead  of  immune  serum) 

171 


172 


TECHNIQUE   OF  THE   COMPLEMENT  FIXATION  METHOD 


-f-  complement  (five  hours)  +  hemolysin  +  blood.  Hemolysis  must  occur,  as  the 
normal  serum  does  not  contain  enough  amboceptors  to  unite  with  the  bacterial  suspen- 
sion and  consequently  complement  remains  unbound. 

2.  Inactivated  immune  serum  +  complement  (five  hours)  +  hemolysin  +  blood. 
Hemolysis  results. 

3.  Inactivated  normal  serum  +  complement  (five  hours)  +  hemolysin  +  blood. 
Hemolysis. 

4.  Antigen  +  inactivated  immune  serum  +  hemolysin  +  blood.     No  hemolysis, 
as  complement  is  absent. 

5.  Antigen  +  inactivated  normal  serum  (five  hours)  +  hemolysin  -f-  blood.     No 
hemolysis,  as  complement  is  absent. 

The  following  is  the  chart  of  the  first  complement  fixation  test  as  originally  per- 
formed by  Bordet  and  Gengou  in  1901  in  which  pest  antibodies  were  demonstrated  in 
the  serum  of  an  immunized  horse. 


Antigen 

Antibodies 

Complement 

Hemolysin  and 
erythrocytes 

Results 

I 

0.4   pest   bacilli 
emulsion. 

''- 
1.2  inactive  pest 
serum  (horse). 

o.  2   guinea-pig's 
serum. 

2  drops  of  rabbit's 
blood  sensitized. 

o 

2 

0.4    pest   bacilli 
emulsion. 

i  .  2  inactive  nor- 
mal serum 
(horse). 

0.2   guinea-pig's 
serum. 

2  drops  of  rabbit's 
blood  sensitized. 

Complete 
hemolysis. 

•i 

i  2  inactive  pest 

o    2  guinea-pig's 

2  drops  of  rabbit's 

Complete 

serum  (horse). 

serum. 

blood  sensitized. 

hemolysis. 

i  2  inactive  nor- 

o 2   guinea-pig's 

2  drops  of  rabbit's 

Complete 

mal  serum 
(horse)  . 

serum. 

blood  sensitized. 

hemolysis. 

5 

0.4    pest   bacilli 
emulsion. 

i  .  2  inactive  pest 
serum  (horse). 

2  drops  of  rabbit's 
blood  sensitized. 

o 

6 

0.4   pest   bacilli 

i  .  2  inactive  nor- 

2 drops  of  rabbit's 

o 

emulsion. 

mal  serum 
(horse). 

blood  sensitized. 

Employing  this  method,  Bordet  and  Gengou  found  positive  results  with  the 
following  combinations: 

1 .  Pest  bacilli  +  pest  horse's  serum  +  guinea-pig  complement  -f-  guinea-pig  hem- 
olysin -t-  rabbit's  blood. 

2.  Anthrax  vaccine  +  guinea-pig  immune  serum  +  guinea-pig  complement  -f- 
guinea-pig  hemolysin  -f  rabbit's  blood. 

3.  Typhoid  bacilli  +  guinea-pig  immune  serum  +  guinea-pig  complement  + 
guinea-pig  hemolysin  +  rabbit's  blood. 

4.  Coli  bacilli  +  guinea-pig  immune  serum  +  guinea-pig  complement  +  guinea- 
pig  hemolysin  +  rabbit's  blood. 

5.  Typhoid  bacilli  -f-  human  convalescent  serum  +  human  complement  +  guinea- 
pig  hemolysin  +  rabbit's  blood. 


TECHNIQUE    OF    WASSERMANN   REACTION  173 

6.  Killed  tubercle  bacilli  -f-  guinea-pig  immune  serum  +  guinea-pig  complement  + 
rabbit's  hemolysiu  +  goat's  blood  or  sheep's  blood. 

7.  Whooping  cough  bacilli  +  patient's  serum  +  guinea-pig's  complement -f  rabbit 
hemolysin  -f  goat's  or  sheep's  blood. 

8.  Meningococci  -+-  human  convalescent's  serum  +  human  complement  +  guinea- 
pig's  hemolysin  +  rabbit's  blood  (Cohen). 

Foix  and  Mallein  examined  twelve  cases  of  scarlet  fever  and  obtained  a 
positive  result  in  ten  cases  when  the  streptococcus  grown  from  a  scarlet 
angina  was  used  as  antigen.  Antibodies  were  found  on  the  fourth  day. 
These  results  were  confirmed  by  Schleissner. 

II.  Wassermann-Bruck's  Modification. 

a.  Antigen. — Instead  of  entire  bacteria,  only  bacterial  extracts  are 
employed.     These  are  made  in  the  same  manner  as  the  artificial  aggressins. 

For  typhoid  bacteria  Leuchs  advises  that  the  bacterial  suspension  should  first  be 
killed  for  twenty-four  hours  at  60°  C.  and  then  shaken  for  two  days.  In  tuberculosis 
good  results  are  obtained  by  using  Koch's  preparation  of  old  and  new  tuberculin. 

The  bacterial  extracts  when  very  fresh  contain  a  great  .deal  of  precipi- 
tinogen  which  diminishes  in  several  days  and  finally  disappears.  Its 
presence  does  not  disturb  complement  fixation.  The  bacterial  extracts 
must  be  well  protected  from  light  and  kept  in  the  cold. 

After  the  extract  has  stood  for  some  time  a  sediment  forms;  under  no  circumstance 
should  this  be  disturbed  or  shaken.  The  required  amount  of  antigen  should  be  care- 
fully poured  off,  and  not  pipetted  off.  Just  as  soon  as  the  required  amount  is  obtained, 
the  extract  should  be  returned  to  the  ice-box. 

b.  The  antiserum  is  inactivated  by  heating,  even  if  the  serum  is  old  and 
contains  very  little  or  no  complement. 

Old,  non-heated  serum  is  often  antihemolytic.  Temperatures  over  60°  C.  should 
be  strictly  guarded  against  as  the  amboceptors  may  be  destroyed.  Heating  for  a 
period, longer  than  one-half  hour  may  make  a  serum  anticomplementary,  i.e.,  bind 
complement.  Sera  containing  bile  at  times  prevent  hemolysis.  Chylous  sera  obtained 
during  the  period  of  digestion  and  milky  sera  seen  in  nursing  women  usually  do  not 
'interfere  with  the  complement  fixation  reaction. 

Exudates,  transudates,  and  spinal  fluids  are  treated  like  sera.  Exu- 
dates  very  rich  in  albumin  frequently  coagulate  during  mactivation. 
In  order  to  avoid  this,  it  is  advisable  to  dilute  such  fluids  with  physio- 
logical salt  solution.  Occasionally  exudates  tend  to  fix  complements 
spontaneously. 


174  TECHNIQUE  OF  THE   COMPLEMENT  FIXATION  METHOD 

c.  Complement  is  obtained  by  killing  a  guinea-pig  and  using  its  serum 
while  fresh.     The  serum  preserved  in  "Frigo"  is,  according  to  Sterns,  not 
reliable. 

d.  Hemolysin  is  represented  by  the  inactivated  serum  of  a  rabbit  that 
has  been  immunized  against  sheep's  red  blood  cells. 

e.  The  twice  washed  sheep's  red  blood  cells  are  used  as  erythrocytes. 

These  five  substances  are  placed  in  the  test-tubes  in  the  following  order:  antigen, 
inactivated  antiserum,  complement;  they  are  thoroughly  mixed  by  shaking  and 
placed  in  the  incubator  for  one  hour  in  order  to  hasten  their  union.  After  this  interval 
the  inactivated  hemolysin  and  the  red  blood  cells  are  added  as  indicator.  The  mixtures 
are  again  returned  to  the  incubator  to  promote  hemolysis.  As  in  all  biological  reac- 
tions, the  quantitative  relationship  of  these  various  ingredients  determines  to  a  great 
extent  the  final  result  of  the  complement  fixation  test.  As  far  as  antigen  and  anti- 
body are  concerned,  the  experiments  of  Weil  and  Nakayama  must  be  considered; 
these  are  to  the  effect  that  only  one-half  of  the  maximum  dose  of  each  ingredient 
which  does  not  bind  complement  is  employed.  With  this  point  in  view,  preliminary 
tests  determining  the  proper  dosage  of  each  must  be  performed. 

The  amount  of  complement  used  is  always  constant.  In  Wassermann's  labora- 
tory i  c.cm.  of  the  dilution  i  :  10  represents  the  quantity  chosen.  For  most  tests  this 
quantity  is  sufficient  as  it  represents  about  three  times  the  titer  of  normal  guinea-pig's 
serum.  In  certain  instances  it  is  preferable  to  work  with  smaller  quantities,  as  in 
Marmorek's  method  of  complement  fixation  with  the  urine  of  tuberculous  patients. 
Of  the  hemolysin  the  two -fold  or  three-fold  titer  dose  is  taken  and  of  the  erythrocytes 
i  c.cm.  of  a  5  per  cent,  suspension  in  normal  saline  solution  suffices.  For  Marmorek's 
test  the  hemolysin  is  employed  in  just  the  titer  dose,  and  of  the  red  blood  cells  only 
0.3  c.cm.  is  taken.  Each  of  the  five  elements  is  diluted  with  saline  to  make  up  i  c.cm. 
so  that  at  the  completion  of  the  test  all  the  tubes  contain  5  c.cm.  Quite  a  difference 
arises  if  an  individual  test  is  performed  with  a  constant  quantity  of  serum  and  dimin- 
ishing doses  of  bacterial  extract  or  reversely.  Important  tests  should  be  carried  out 
by  both  methods.  The  necessary  controls  are: 

1.  The  double  dose  of  antigen  +  complement  -f  hemolysin  -f  blood,  to  prove  that 
the  dose  of  antigen  employed  in  the  test  is  correct  (Weil  and  Nakayama). 

2.  The  double  quantity  of  serum  +  complement  +  hemolysin  -f-  blood,  to  prove 
that  the  dose  of  serum  employed  is  correct  (Weil  and  Nakayama). 

3.  The  "system  control";  blood  -f  complement  +  one-half  amount  of  hemolysin, 
to  show  that  the  test  was  performed  with  double  the  hemolytic  dose. 

4.  Blood  +  salt  solution,  to  prove  that  the  salt  solution  is  isotonic. 

In  addition,  it  is  advisable  to  repeat  the  test  with  inactivated  normal  serum  sub- 
stituted for  the  immune  serum  and  another  with  a  foreign  instead  of  a  homologous 
antigen. 

These  controls  assure  beyond  doubt  the  specificity  of  the  reaction. 

The  accompanying  chart  represents  schematically  all  that  has  been  discussed. 


ESTIMATION   OF    STRENGTH   OF   MENINGOCOCCUS    SERUM 


Titration  of  a  Meningococcus  Serum  Obtained  from  the  Horse, 
a.  Diminishing  Quantities  of  Antigen. 


Antigen 

Antibodies 

Complement 

Hemolysin 

Erythrocytes 

:  •,;..;. 

Results 

0.25      c.cm. 

o  .  I  c.c.  inactive 

o.i  c.cm. 

0.002  c.cm.  in- 

I c.cm.  5  % 

. 

Meningococcus 

immune  serum 

guinea-pig's 

active  rabbit's 

sheep's  blood 

_^__  .. 
o  hemolysis 

extract 

serum 

(sheep)  serum 

o.i        c.cm. 

" 

" 

" 

" 

o  hemolysis 

0.05       c.cm. 

" 

" 

" 

o  hemolysis 

o.oi       c.cm. 

" 

" 

" 

" 

o  hemolysis 

0.005    c.cm. 

" 

" 

« 

" 

Incomplete  hemolysis 

o.ooi    c.cm. 

" 

" 

" 

" 

Incomplete  hemolysis 

0.0005  c.cm. 

" 

" 

" 

" 

Complete  hemolysis 

i.o    c.cm. 

M 

,< 

,i 

Incomplete  hemolysis 

0.5    c.cm. 

H 

., 

.1 

Complete  hemolysis 

o.  25  c.cm. 

M 

M 

H 

Complete  hemolysis 

Antigen 

Antibodies 

Complement 

Hemolysin 

Erythrocytes 

Result 

Inactive  immune 

serum. 
0.4  c.cm. 

'• 

» 

" 

Incomplete  hemolysis 
Complete  hemolysis 

o.  i  c.cm. 

« 

« 

« 

Complete  hemolysis 

„ 

o  ooi  c.cm. 

„ 

Complete  hemolysis 

.1 

o  hemolysis 

M  eningococcus 
extract. 
0.25  c.cm. 
o.i    c.cm. 

Inactive  normal 
horse's  serum, 
o.  i  c.cm. 
o.  i  c.cm. 
o  2  c  cm. 

,, 

0.002  c.cm. 

Almost  complete 
hemolysis 

Complete  hemolysis 
Complete  hemolysis 

Staphylococcus 
extract 
0.25  c.cm. 
o.i    c.cm. 
0.5    c.cm. 

Inactive  menin- 
gococcus  serum 
o.  i  c.cm. 
o  .  i  c.cm. 

« 

., 

Complete  hemolysis 

Complete  hemolysis 
Complete  hemolysis 

The  titer  of  the  meningococcus  serum  is  o.ooi  c.cm.  of  antigen.  Since  i.o  c.cm.  of 
antigen  binds  o.i  complement  and  0.5  c.cm.  does  not  interfere  with  hemolysis,  the 
maximum  dose  of  antigen  which  may  be  used  for  the  trial  is  0.25  c.cm. 

Inasmuch  as  0.4  c.cm.  of  the  inactivated  serum  binds  a  part  of  the  complement,  and 
0.2  does  not  at  all  interfere  with  hemolysis,  the  maximum  dose  of  serum  to  be  employed 
is  o.i  c.cm. 

The  positive  reaction  must  be  attributed  to  the  interaction  between  antigen  and 
antibody,  as  hardly  any  complement  fixation  takes  place  by  using  inactivated  normal 
serum  with  0.25  c.cm.  of  antigen.  That  the  reaction  is  specific  is  shown  by  hemolysis 
occurring  when  the  homologous  antigen  is  substituted  by  a  Staphylococcus  extract. 


176  TECHNIQUE   OF  THE   COMPLEMENT  FIXATION  METHOD 

b.  Same  with  Diminishing  Amounts  oj  Serum. 


Antigen 

Antibodies 

Complement 

Hemolysin 

- 
Erythrocytes 

Result 

o.  25  c.cm.  men- 

o.  i  c.cm.  inac- 

o. i  c.cm. 

0.002  c.cm. 

i  c.cm.  5  % 

o  hemolysis 

ingococcus  ex-. 

tive  meningo- 

guinea-pig's 

sheep's  blocd. 

tract. 

coccus  serum. 

serum. 

•• 

0.05    c.cm. 

" 

0.002  c.cm. 

" 

o  hemolysis 

" 

o.oi    c.cm. 

" 

0.002  c.cm. 

" 

0  hemolysis 

•• 

0.005  c.cm. 

" 

0.002  c.cm. 

" 

o  hemolysis 

" 

o.ooi  c.cm. 

" 

0.002  c.cm. 

•f 

Almost  o  hemolysis 

" 

0.0005  c.cm. 

" 

0.002  c.cm. 

" 

Incomplete  hemolysis 

" 

o.oooi  c.cm. 

*' 

o.  002  c.cm. 

" 

Complete  hemolysis 

0.5  c.cm.  men- 

,, 

o.  002  c.cm. 

,, 

Complete  hemolysis 

ingococcus  ex- 

tract. 

Antigen 

Antibodies 

Complement 

Hemolysin 

Erythrocytes 

Results 

o  2  c.cm. 

„ 

.. 

Comp1ete  hemolvsis 

,, 

o  ooi  c  cm 

,, 

Complete  hemolysis 

„ 

o  hemolysis 

0.25  c.cm.  men- 
ingccoccus  ex- 
tract. 

Inactive  normal 
horse's  serum. 
o.i    c.cm. 
0.05  c.cm. 

,, 

o.  002  c.cm. 
0.002  c.cm. 

,4 

Almost  complete 
hemolys.s 

Complete  hemolysis 

0.2    c.cm. 

¥ 

0.002  c.cm. 

" 

Complete  hemolysis 

0.25  c.cm.  sta- 
phylococcus  ex- 
tract. 

o.i  c.c.  inactive 
meningococcus 

serum. 

0.002  c.cm. 

" 

Complete  hemolysis 

o.  5  c.cm.  sta- 

., 

o  002  c.cm. 

Complete  hemolysis 

phylococcus  ex- 
tract. 

With  0.25  c.cm.  of  antigen  the  titer  of  this  meningococcus  serum  is  0.0005  c.cm.  of 
serum.  One  could  with  this  constant  quantity  of  serum,  and  varying  quantities  of 
antigen,  titrate  the  minimum  amount  of  antigen  necessary  for  complement  fixation. 
It  would  even  be  preferable  for  such  a  test  to  employ  0.005  c-cm-  of tne  serum,  as  this 
amount  surely  binds  no  complement.  If  such  a  titration  is  undertaken  it  will  be  found 
that  0.005  c-cm-  of  serum  with  0.05  c.cm.  of  extract  can  bind  o.i  c.cm.  of  complement. 

Similarly  the  antibodies  contained  in  the  blood  serum  or  spinal  fluid  of  a 
patient  can  be  determined  by  means  of  complement  fixation. 

If  it  is  desired  to  demonstrate  the  antigen  instead  of  antibody,  one  pro- 
ceeds as  follows: 


ANTIGEN  PROPERTIES  OF  CEREBROSPINAL  FLUID 


I77 


c.  Demonstration  of  meningococcus  antigen  in  the  spinal  fluid  of  a  patient 
•with  a  possible  meningitis. 


Antigen 

Antibodies 

Complement 

Hemolysin 

Blood 

Results 

0.5  c.cm.  active 
spinal  fluid  from 
patient. 
0.3  c.cm. 
o.  i  c.cm. 

o.i  c.cm.  inactive 
horse's   meningo- 
coccus serum. 
o.  i  c.cm. 
o.  i  c.cm. 

o.  i  c.cm. 

o.  i  c.cm. 
o.  i  c.cm. 

0.002  c.cm. 

0.002  c.cm. 
0.002  c.cm. 

i  c.cm.  5  % 

i  c.cm.  5% 
i  c.cm.  5% 

o  hemolysis 

Incomplete  hemolysis 
Complete  hemolysis 

o.  2  c.cm. 

o.  i  c.cm. 

o.  002  c.cm. 

i  c.cm.  5  % 

Complete  hemolysis 

I  o  c.cm. 

o  i  c.cm. 

o  002  c  cm. 

i  c.cm.  5  % 

Almost  complete 

0.6  c.cm. 

o.  i  c.cm. 

0.002  c.cm. 

i  c.cm.  5  % 

hemolysis 
Complete  hemolysis 

o.  i  c.cm. 

o.ooi  c.cm. 

i  c.cm.  5  % 
i  c.cm.  5  % 

Complete  hemolysis 
o  hemolysis 

0.5  c.cm.  active 
normal  spinal 
fluid. 
I  o  c.cm. 

o.  i  c.cm. 

o.  i  c.cm. 
o  i  c.cm. 

0.002  c.cm. 
o  002  c.cm. 

i  c.cm.  5  % 
i  c.cm.  5  % 

Complete  hemolysis 
Complete  hemolysis 

0.5  c.cm.  of  ac- 
tive spinal  fluid 
from  patient. 
0.3  c.cm. 

o.  i  c.cm.  inac- 
tive normal 
horse's  serum. 

o.  i  c.cm. 
o.  i  c.cm. 

0.002  c.cm. 
0.002  c.cm. 

i  c.cm.  5  % 
i  c.cm.  5% 

Almost  complete 
hemolysis 

Complete  hemolysis 

0.05  c.cm.  men- 
ingococcus ex- 
tract. 

0.005  c.cm.  inac- 
tive meningococ- 
cus serum. 

o.  i  c.cm. 

0.002  c.cm. 

i  c.cm.  5% 

o  hemolysis 
Look  at  previous  test 

The  spinal  fluid  contains  meningococcus  antigen  thus  proving  that  the  patient  is 
suffering  from  epidemic  cerebrospinal  meningitis.  Neither  the  double  amount,  of 
serum,  a  double  amount  of  antigen,  a  mixture  of  normal  spinal  fluid  with  specific  serum, 
nor  normal  serum  with  the  specific  spinal  fluid  binds  complement.  Only  a  mixture  of 
meningococcus  extract  and  specific  serum  gives  complement  fixation. 

The  results  obtained  by  complement  fixation  depend  to  a  great  extent 
upon  the  quantitative  relationship  of  the  various  ingredients.  The  affinity 
toward  the  complement  existing  between  the  antigen  and  amboceptor  on 
the  one  hand,  is  balanced  by  that  between  the  hemolysin  +  blood  on  the 
other.  By  modifying  their  quantitative  proportions  different  results  may 
be  obtained.  If  for  example  the  strength  of  the  hemolysin  is  excessively 
increased,  it  is  possible  that  the  previously  bound  complement  is  again  de- 
tached and  hemolysis  ensues.  Originally  the  results  were  read  after  the 
mixtures  had  remained  two  hours  in  the  incubator  and  twenty-four  hours 
in  the  ice-box.  At  the  present,  most  authorities  agree  to  read  the  results 
at  a  time  when  the  control  tubes  are  ready;  that  is  when  the  complement 
is  bound  or  hemolysis  has  been  completed  in  those  tubes  in  which  these 
respective  phenomena  should  occur. 


12 


178  TECHNIQUE  OF  THE   COMPLEMENT  FIXATION  METHOD 

Wassermann's  modification  of  the  Bordet-Gengou  method  was  first 
practically  employed  for  the  titration  of  the  therapeutic  meningococcus. 
serum.  One  cannot,  however,  correctly  judge  the  prophylactic  or  curative 
value  of  a  serum  by  its  antibody  content  as  they  do  not  run  hand  in  hand 
(R.  Kraus,  F.  Meyer  and  Garbat,  Citron.) 

ffl.  Serum  Diagnosis  of  Syphilis. 

a.  Wassermann's  Technique. 

The  technique  of  this  reaction  as  carried  out  in  Wassermann's  labora- 
tory is  practically  identical  with  that  just  described  for  the  diagnosis*of 
bacterial  infections.  The  preparation  of  the  antigen  varies  slightly. 

The  liver  obtained  from  a  syphilitic  fetus  is  weighed  and  cut  up  into  fine  pieces. 
Four  times  its  weight  of  1/2  per  cent,  of  carbolic  solution  in  saline  is  added,  the  mix- 
ture placed  in  a  brown  bottle  and  shaken  for  twenty-four  hours.  It  is  then  cen- 
trifugalized  until  the  larger  liver  remnants  settle  to  the  bottom  and  a  somewhat  turbid 
fluid  remains  above.  The  latter  is  poured  off  into  a  brown  bottle  and  placed  in  the 
ice-box.  After  several  days  of  sedimentation,  the  fluid  assumes  a  yellowish-brown 
opalescence  and  can  now  be  used  as  a  luetic  antigen.  It  should  not  be  exposed  to  light 
or  heat,  should  not  be  shaken,  and  its  contents  should  not  be  pipetted  off,  but  care- 
fully poured  off  without  disturbance  to  the  sediment. 

By  titration  of  the  extract,  that  dose  is  determined  which  does  not  of 
itself  bind  complement.  Only  such  extracts  are  kept  which  in  the  dose  of 
0.4  c.cm.  do  not  interfere  with  hemolysis. 

Control  tests  should  also  be  made  to  ascertain  whether  the  organ  extract 
has  any  tendency  of  its  own  to  hemolyze  red  blood  cells  without  the 
presence  of  complement  or  hemolysin. 

Not  every  luetic  extract  can  serve  as  antigen  for  complement  fixation. 
A  number  of  other  substances,  both  normal  and  pathological,  may  be 
extracted  from  the  luetic  liver  besides  that  agent  necessary  for  the 
Wassermann  test.  These  undesired  ingredients  may  interfere  with  the 
efficiency  of  the  extract.  For  this  reason  a  great  number  of  known  posi- 
tive and  negative  sera  should  be  tested  with  each  new  extract,  and  only 
if  the  results  are  absolutely  correct  should  it  be  employed  as  antigen. 

In  the  early  work  of  Wassermann  the  antigen  was  described  as  deteriorating  very 
easily;  its  activity  would  either  be  entirely  destroyed  or  it  would  become  anticomple- 
mentary.  The  author  is  firmly  convinced  that  these  changes  are  brought  about  by 
careless  handling  of  the  extract  or  its  exposure  to  light.  If  properly  taken  care  of,  its 
activity  remains  constant. 

From  practical  experience,  it  has  been  found  that  extracts  which  must 
be  used  in  amounts  less  than  o.i  c.cm.  are  as  a  general  rule  unsatisfactory. 


TITRATION   OF   ANTIGEN 


179 


Similarly,  the  luetic  sera  are  most  active  when  doses  of  0.2  and  o.i  c.cm. 
are  employed.  Amounts  greater  than  0.2  may  result  in  an  unspecific 
reaction.  The  most  favorable  combinations  are, 

0.2  c.cm.  of  extract  +  0.2  c.cm.  serum. 

o.i  c.cm.  of  extract  +  o.i  c.cm.  serum. 
The  accompanying  table  presents  the  titration  of  an  antigen  in  detail. 

a.  Preliminary  Test — Titration  of  the  Antigen. 


Com- 

Antigen 

ple- 

Hemolysin 

Blood 

Result 

ment 

o.  8  c.cm.  luetic  extract 

0.  I 

Twice  the  hemolytic  dose. 

i  c.cm.  5% 

Incomplete  hemolysis. 

o.  6  c.cm.  luetic  extract 

O.  I 

Twice  the  hemolytic  dose. 

i  c.cm.  5% 

o 

0.4  c.cm.  luetic  extract 

O.  I 

Twice  the  hemolytic  dose. 

i  c.cm.  5% 

Complete  hemolysis. 

o.  2  c.cm.  luetic  extract 

V 

Twice  the  hemolytic  dose. 

i  c.cm.  5% 

Complete  hemolysis. 

0.8  c.cm.  luetic  extract 

i  c  cm    ">% 

Incomplete 

0.6  c.cm.  luetic  extract 

i  c.cm.  5% 

o 

0.4  c.cm.  luetic  extract 

i  c  cm   5% 

o 

0.2  c.cm.  luetic  extract 

i  c  cm    ^  % 

o 

The  test  proves  that  0.4  c.cm.  of  extract  is  not  able  to  bind  o.i  c.cm.  of 
complement.  That  0.8  c.cm.  of  lues  extract  causes  only  an  incomplete 
hemolysis,  while  0.6  c.cm.  produces  no  hemolysis  whatever,  is  explained 
not  by  its  lessened  tendency  of  binding  complement,  but  by  the  greater 
amount  of  hemotoxin  which  0.8  c.cm.  possesses. 

Keeping  in  mind  the  rule  of  Weil  and  Nakayama  about  the  summa- 
tion of  antigen,  this  particular  antigen  will  be  employed  in  the  quantities 
of  0.2  c.cm.  and  o.i  c.cm.  (0.2  being  1/2  of  0.4  c.cm.  which  is  the  largest 
amount  that  does  not  of  itself  fix  complement) . 

b.  Examination  and  titration  of  4  luetic  sera  by  Citron's  method  (see 
Plate  II). 

The  technical  details  of  the  test  are  as  follows: 

Three  test-tubes  are  assigned  for  each  test  and  placed  in  a  test-tube  rack.  The 
name  of  the  patient  is  written  upon  the  first  of  these  tubes.  Another  rack  contains  one 
tube  for  each  patient  and  labelled  accordingly.  In  addition  there  is  an  "  antigen  tube," 
which  is  placed  in  the  first  rack  at  the  end  of  all  the  other  tubes;  also  a  "normal 
extract,"  a  "system,"  "complement,"  and  "blood  control  tube,"  which  are  placed 
in  the  second  rack.  The  amount  of  syphilitic  antigen  required  for  the  entire  work 
is  calculated  as  follows.  For  each  test  0.3  of  antigen  is  required;  for  five  cases  (in- 
cluding controls)  1.5  c.cm.  are  needed  +  0.4  c.cm.  for  the  antigen  control  tube  =1.9 
c.cm.  in  all,  or  2.0  c.cm.  in  round  numbers.  This  amount  is  diluted  with  normal  salt 
solution  in  the  proportion  of  i  :  5  so  that  8  c.cm.  of  saline  are  added  (  —  2  :  10) ;  i  c.cm. 
of  this  dilution  contains  0.2  antigen  and  1/2  c.cm.  contains  o.i  antigen.  The  first 
tube  (i,  4,  7,  ic,  etc.,  in  diagram)  of  every  test  therefore  receives  i  c.cm.,  the  second 


i8o 


TECHNIQUE   OF  THE   COMPLEMENT  FIXATION  METHOD 


tube  1/2  c.cm<,  the  third  tube  nothing,  the  antigen  tube  (tube  19)  2  c.cm.  Physiolog- 
ical salt  solution  is  added  to  make  up  i  c.cm.  in  each  tube;  first  tube  nothing;  second 
tube  1/2  c.cm.;  third  tube  i  c.cm.  of  saline. 

The  normal  extract  required  for  the  tubes  in  the  second  rack  is  similarly  estimated, 
0.2  c.cm.  is  needed  for  each  test,  5  (tests)  Xo.2  =  1.0+0.4  fc>r  the  antigen  control  tubes 
=  1.4  or  1.5  c.cm.  in  round  numbers.  For  purposes  of  dilution  i  :  5,  6  c.cm.  of  salt 
solution  are  added  and  i  c.cm.  (  =  0.2  of  extract)  placed  into  each  of  the  tubes  (20  to 
26)  and  2  c.cm.  into  the  normal  extract  control  test-tube  (tube  27).  In  this  series 
also,  salt  solution  is  added  to  make  up  equal  quantities  of  i  c.cm. :  first  tube  nothing, 
second  tube  1/2  c.cm.,  third  tube  i  c.cm.;  antigen  tube  (27),  nothing;  system  (28), 
complement  (29)  and  blood  (30)  control  tubes  each  i  c.cm. 

The  second  ingredient  of  the  test  is  next  added,  i.e.,  the  respective  serum.  This  is 
not  diluted  but  added  directly;  0.2  c.cm.  into  the  first  tube  of  each  test;  o.i  c.cm.  into 
the  second;  0.2  c.cm.  into  the  third  tube;  also  0.2  into  the  control  series  of  tubes  labelled 
with  the  patient's  names  in  the  second  rack.  Salt  solution  is  again  added  to  make 
up  to  the  equal  quantity  of  2  c.cm.  in  each  tube,  thus:  0.8  c.cm.  into  first,  0.9  c.cm.  into 
the  second,  0.8  c.cm.  into  the  third  tube,  and  0.8  c.cm.  into  the  control  series;  nothing 
into  the  antigen  tubes,  i  c.cm.  into  system,  complement,  and  blood  control  tubes. 

The  addition  of  complement  follows  next.  Each  tube,  except  the  blood  tube, 
receives  o.i  c.cm.  of  complement.  Thus  the  tubes  are  counted  and  if,  for  example, 
nineteen  tubes  are  present  19X0.1  c.cm.  complement  is  taken,  or  in  round  numbers 
2  c.cm. 

Complement  is  always  diluted  i  :  10,  or  2  c.cm.  complement  +  18  c.cm.  saline,  so 
that  each  tube  except  the  blood  tube  (30)  receives  i  c.cm.  of  this  diluted  complement. 
Tube  (30)  receives  i  c.cm.  of  saline  instead.  All  tubes  are  then  carefully  shaken  and 
the  racks  placed  in  the  incubator  for  one  hour. 

During  this  time,  the  hemolysin  and  washed  red  blood  cells  are  properly  diluted. 
The  red  blood  cells  are  made  up  in  a  5  per  cent,  suspension  of  which  each  tube  will 
receive  i  c.cm.  Thus  in  the  present  test  there  are  thirty  tubes,  requiring  30  c.cm.  of 
blood  suspension;  since  i  c.cm.  of  washed  blood  when  diluted  i  :  20  will  supply  twenty 
tubes,  for  30  c.cm.  about  i  1/2  c.cm.  of  blood  will  be  required,  or  2  c.cm.  will  make 
40  c.cm.  of  a  5  per  cent,  blood  suspension. 


Luetic 
extract 

Serum. 

Comple- 
ment 

Hemoly- 
sin;ic.cm. 

Sheep's 
blood 

Result  of  hemolysis 

I.     O.  2 

o.  2    Ser.  I.  Tabes   un- 

0. I 

i  :  looo 

i  c.cm.  5% 

No  hemol- 

treated; without  luetic 

ysis. 

history. 

2.     O.  I 

o.  i  As  above. 

0.  I 

i  :  looo 

i  c.cm.  5% 

No  hemol- 

'  4-  +  +  + 

ysis. 

3- 

0.21  As  above. 

O.  I 

i  :  1000 

i  c.cm.  5% 

Complete 

I 

hemolysis. 

4.     0.  2 

0.2  Ser  II.  Secondary 

O.  I 

i  :  1000 

i  c.cm.  5% 

No  hemol- 

lues untreated. 

ysis. 

5-    o.i 

o.  i  As  above. 

O.  I 

i  :  looo 

i  c.cm.  5% 

Incomplete 

hemolysis. 

'       ' 

6.    ... 

o.  21  As  above. 

0.  I 

i  :  1000 

i  c.cm.  5% 

Complete 

hemolysis.    , 

TITRATION  OF  LUETIC  SERA 


181 


Luetic 
extract 

Serum 

Comple- 
ment 

Hemoly- 
sin;  ic.cm. 

Sheep's 
blood. 

Result  of  hemolysis 

7.    0.2 

0.2    Ser.    Ill    Tabes. 

O.  I 

i  :  looo     i  c.cm.  5% 

No  hemoly- 

Many inunction  courses 

sis. 

8.    o.i 

o.  i  As  above. 

0.  I 

i  :  looo 

i  c.cm.  5% 

Complete 

•  4-4- 

hemolysis. 

\     \ 

9.    ... 

o.  2  As  above. 

O.  I 

i  :  looo 

ic.  cm.  5% 

Complete 

hemolysis. 

IO.     O.2 

0.2  Ser.  IV  Gallstones. 

0.  I 

i  :  looo 

i  c.cm.  5% 

Trace  of  bind- 

Lues seventeen  years  ago. 

ing;  almost  but 

Much    treatment.     No 

not  quite  com- 

symptoms for  ten  years. 

plete  hemolysis. 

4- 

II.     O.I 

o.i  As  above. 

O.I 

i  :  looo 

i  c.cm.  5% 

Complete 

hemolysis. 

12.      ... 

o.  2  l  As  above. 

O.I 

i  :  looo 

i  c.cm.  5% 

Complete 

hemolysis. 

13.     0.2 

0.2    Negative    control 

0.  I 

i  :  looo 

i  c.cm.  5% 

Complete 

| 

serum          (carcinoma 

hemolysis. 

hepatis). 

•  — 

14-      2.J 

0.2  l  As  above. 

0.  I 

i  :  1000 

ic.  cm.  5% 

Complete 

hemolysis. 

' 

15.    o.i 

o.  i    Strongly    positive 

0.  I 

i  :  1000 

i  c.cm.  5% 

No  hemol- 

control serum.     (Lues 

ysis. 

maligna). 

•  +  +  -h  + 

16.    ... 

o.  2  As  above. 

O.  I 

i  :  1000 

i  c.cm.  5% 

Complete 

hemolysis. 

17.     0.2 

o.  2  Weakly  positive  con- 

O. I 

i  :  looo 

i  c.cm.  5% 

Incomplete 

trol    serum.     Primary 

hemolysis. 

lesion. 

\  j. 

18.    ... 

0.21    Weakly    positive 

O.  I 

i  :  1000 

i  c.cm.  5% 

Complete 

i 

control    serum.     Pri- 

hemolysis. 

mary  lesion. 

10.    0.4 

O.  I 

i  :  1000 

i  c.cm.  5% 

Complete  hemolysis. 

V  •          v*  T^ 

Normal 

extract. 

20.    0.2 

o.  2  Serum  I. 

O.I 

IOOO 

i  c.cm.  5% 

Complete  hemoiysis. 

21.     0.2 

o.  2  Serum  II. 

O.  I 

IOOO 

i  c.cm.  5% 

Incomplete  hemolysis, 

22.     O,2 

o.  2  Serum  III. 

O.I 

IOOO 

i  c.cm.  5% 

Complete  hemolysis. 

23.     0.2 

o.  2  Serum  IV. 

O.I 

IOOO 

i  c.cm.  5% 

Complete  hemolysis. 

24.     0.2 

0.2    Negative    control 

O.  I 

IOOO 

i  c.cm.  5% 

Complete  hemolysis. 

serum. 

25.     0.2 

0.2    Strongly    positive 

O.I 

I       IOOO 

i  c.cm.  5% 

Complete  hemolys  . 

control  serum. 

26.     0.2 

0.2     Weakly     positive 

O.  I 

I       IOOO 

i  c.cm.  5% 

Complete  hemolysis. 

control  serum. 

27.     0.4 

O.  I 

I    1  IOOO 

i  c.cm.  5% 

Complete  hemolysis. 

28.      ... 

O.  I 

i  :  1000 

i  c.cm.  <?% 

Complete  hemolysis. 

29.      ... 

O    I 

J.     V«.VXJ.X.    ^    /Q 

i  c.cm.  5% 

No  hemolysis. 

30.      ... 

i  c.cm.  5% 

No  hemolysis. 

182 


TECHNIQUE   OF   THE   COMPLEMENT  FIXATION  METHOD 


The  hemolysin  is  prepared  as  follows:  its  titer  for  example  is  i  :  2000  and  it  is  em- 
ployed in  the  dilution  of  i  :  1000.  Each  tube  except  the  blood  and  complement  tubes 
will  receive  i  c.cm.  of  the  diluted  hemolysin;  i  c.cm.  of  the  latter  if  diluted  properly 
would  give  1000  c.cm.;  o.i  of  the  hemolysin  which  is  the  smallest  amount  that  can  be 
measured  will  give  100  c.cm.  Every  tube  except  28  to  30  will  receive  i  c.cm.  of  the 
hemolysin  dilution  i  :  1000.  Tubes  29  and  30  will  receive  none  (replaced  by  saline), 
tube  28  will  receive  1/2  c.cm.  of  the  hemolysin  and  1/2  c.cm.  of  saline. 

After  an  hour's  incubation,  each  tube  receives  i  c.cm.  of  R.  B.  C.  and  i  c.cm.  of 
the  hemolysin  just  mentioned.  If  it  is  desired  to  hasten  the  results,  it  is  advisable 
to  mix  a  sufficient  equal  quantity  of  R.  B.  C.  and  hemolysin  solution  (30  c.cm.  of  each) 
and  allow  the  mixture  to  remain  in  the  incubator  for  a  short  time  before  the  hour's 
incubation  is  up.  Then  instead  of  adding  i  c.cm.  of  these  ingredients  separately, 
2  c.cm.  of  the  mixture  is  added  to  all  except  tubes  28  to  30.  Tubes  29  and  30  receive 
i  c.cm.  of  blood  and  i  c.cm.  of  saline  and  tube  28  i  c.cm.  of  blood,  1/2  c.cm.  of 
hemolysin  and  1/2  c.cm.  of  saline. 

Meier's  Modification. 

For  over  a  year  G.  Meier  in  Wassermann's  laboratory  has  been  using  a  turbid  sus- 
pension of  the  syphilitic  liver  instead  of  the  clear  antigen  as  prepared  above.  After 
the  liver  is  cut  up  into  fine  pieces,  placed  into  four  times  its  weight  of  one-half  per  cent, 
carbolic  acid  solution  in  normal  saline  and  shaken  for  24  hours,  the  mixture  is  filtered 

I  (Qualitative  Test) 


A  known 
positive  serum 

A  known 
negative  serum 

Salt  solution 

Extract  X 

O    2 

Complete 

Few  non-hem- 

Few  non-hem- 

fixation. 

olyzed  red  cells. 

olyzed  red  cells. 

Extract  X  

O    I 

Complete 

Hemolysis. 

Hemolysis. 

fixation. 

Extract  X 

O   O^ 

Complete 

Hemolysis 

Hemolysis 

fixation. 

Extract  X 

O    O2  ^ 

Complete 

Hemolysis 

Hemolysis 

fixation. 

Extract  X  .  . 

o  01  25 

Incomplete 

Hemolysis 

Hemolysis 

hemolysis. 

Known  syphilitic  extract  

O    O3S 

Complete 

Hemolysis  . 

(o  07} 

fixation. 

Hemolysis. 

Salt  solution  with  double  the  quan- 

Hemolysis. 

Hemolysis. 

tity  of  serum. 

1  o.  4  c.cm.  of  luetic  serum  frequently  binds  complement  of  its  own  accord.  Experience  has 
shown  that  if  o.  2  c.cm.  does  not  bind  complement  and  o.  2  c.cm.  of  serum  +  0.2  c.cm.  of  anti- 
gen does  bind  complement,  the  unknown  serum  is  surely  of  luetic  origin. 


MEIER  S    MODIFICATION    OF   THE    WASSERMANN   TEST 


through  gauze  to  remove  the  very  large  particles.  The  filtrate  is  then  ready  for  use. 
It  is  light  or  dark  brown  in  color,  and  translucent  even  in  weak  dilution.  On  standing, 
a  brown  sediment  settles  to  the  bottom;  before  being  used  the  extract  should  be 
thoroughly  mixed  as  in  this  way  it  is  five  to  ten  times  stronger  than  if  the  supernatant 
clear  fluid  alone  is  taken. 

The  efficiency  of  the  extract  is  proven  in  two  ways:  i.  The  orientation  or  qualita- 
tive test,  to  determine  whether  the  new  extract  is  specific  for  lues.  2.  The  quantitative 
test,  to  determine  the  exact  dosage  necessary  for  complement  fixation.  (See  Table  I.) 

Thus  it  is  observed  that  for  both  of  these  standard  sera  (positive  and  negative) 
this  extract  can  be  employed  in  two  doses  0.05,  0.025. 

The  next  step  is  to  determine  which  of  these  two  doses  is  the  more  efficient  for  the 
greatest  number  of  sera.  The  largest  quantity  of  antigen  is  always  preferred;  but  it  is 
a  known  fact  that  the  optimum  for  one  serum  is  not  equally  that  for  another  serum. 
Accordingly,  parallel  examinations  are  made  of  a  great  number  of  sera,  employing 
both  the  new  antigen  and  the  old  control  one,  which  has  already  been  tested  out  and 

II  (Quantitative  Test) 


Rack  I  with  standard 
extract 

Rack  II  with  new 
extract,  dose  0.025 

Rack  III.     Salt  solu- 
tion with  double  the 
quantity  of  serum 

i.  Serum  A  

Complete  hemolysis. 

Complete  hemolysis. 

Complete  hemolysis. 

2.  Serum.  B   

No  hemolysis. 

No  hemolysis. 

Complete  hemolysis. 

3.  Serum  C  

Incomplete  hemolysis. 

Complete  hemolysis. 

Complete  hemolysis. 

4.  Serum  D  

Complete  hemolysis. 

Complete  hemolysis. 

Complete  hemolysis. 

5.  Serum  E  

No  hemolysis. 

Moderate   hemolysis. 

Complete  hemolysis. 

6.  Serum  F  

No  hemolysis. 

Good  deal  of  hemol- 

Complete hemolysis. 

ysis. 

7.  Serum  G  

Almost  complete 

Complete  hemolysis. 

Complete  hemolysis. 

hemolysis. 

8.  Serum  H.. 

Complete  hemolysis. 

Complete  hemolysis. 

Complete  hemolysis. 

'•       *."  •- 

o    Serum  I 

No  hemolysis 

No  hemolysis. 

Complete  hemolysis. 

10.  Serum  K. 

No  hemolysis. 

Very  slight  hemolysis. 

Complete  hemolysis. 

ii.  Known    luetic 
serum. 

No  hemolysis. 

Very  slight  hemolysis. 

Complete  hemolysis. 

12.  Known    normal 
serum. 

Hemolysis. 

Hemolysis. 

Complete  hemolysis. 

13.  Salt  solution  with 
double  the  quantity 
of  extract. 

Hemolysis. 

Hemolysis. 

Complete  hemolysis. 

184  TECHNIQUE  OF  THE  COMPLEMENT  FIXATION  METHOD 

found  to  give  absolutely  reliable  results;  i.e.,  negative  reactions  with  non-luetic 
bloods,  and  a  high  percentage  of  positive  results  (almost  100  per  cent.)  in  cases  with 
florid  non-treated  lues.  If  the  new  extract  is  found  to  give  a  negative  or  weakly  posi- 
tive test  while  the  standard  extract  shows  a  strongly  positive  reaction,  it  is  evident 
that  the  dose  of  the  new  antigen  must  be  increased;  vice  versa,  if  the  new  extract 
gives  too  many  positive  results  especially  with  sera  of  known  non-luetic  origin,  the  dose 
should  be  diminished.  (See  Table  II.) 

From  this  table  it  is  readily  seen  that  the  new  extract  conforms  fully  in  its  results 
with  the  standard  extract  as  far  as  the  negative  sera  and  the  two  strongly  positive  (2 
and  9)  sera  are  concerned.  In  other  cases  (5,  6  and  10)  the  new  extract  gives  only  par- 
tial inhibition  of  hemolysis  while  with  the  control  antigen  complete  inhibition  results. 
With  sera  3  and  7  there  is  complete  hemolysis  instead  of  partial  fixation.  These  data 
prove  that  the  new  antigen  is  not  sufficiently  active  in  the  dose  of  0.025;  consequently 
another  set  of  control  reactions  must  be  undertaken  this  time  employing  0.05  of  the 
antigen.  By  thus  repeatedly  altering  the  dose  cf  the  antigen,  the  results  will  finally 
tally  approximately  with  the  standard  extract  (rarely  do  they  do  so  absolutely)  and 
only  then  may  the  former  be  used  for  the  routine  examinations.  Once  the  proper 
dose  of  this  watery  extract  is  established,  it  remains  constant  for  a  very  long  period. 
If  the  strength  of  the  antigen  changes  at  all,  it  does  so,  in  contrast  to  the  alcoholic 
extracts  of  normal  organs,  only  very  gradually,  and  may  then  have  to  be  used  in 
smaller  or  greater  dosage  as  determined  by  renewed  tit  ration. 

Several  sera  from  cases  with  high  temperatures  or  scarlet  fever  should  be  included 
among  the  tests  as  in  these  conditions  the  tendency  toward  inhibition  of  hemolysis 
in  the  presence  of  lipoids  is  increased. 

For  the  sake  of  economy,  Meier  has  been  working  with  one-half  quantities  of  all 
the  ingredients;  thus 

0.5  c.cm.  of  the  diluted  antigen instead  of  i  .o  c.cm. 

o.i  c.cm.  of  the  diluted  serum instead  of  o.  2  c.cm. 

0.5  c.cm.  of  the  diluted  complement instead  of  i  .o  c.cm. 

0.5  c.cm.  of  the  diluted  hemolysin instead  of  i  .o  c.cm. 

0.5  c.cm.  of  the  diluted  red  cell  emulsion. .  .instead  of  i  .o  c.cm. 

The  hemolysin  is  taken  in  three  to  four  times  its  minimum  hemolytic  dose  or  titer, 
as  the  turbid  antigen  per  se  inhibits  hemolysis  more  strongly  than  the  clear  extracts. 
In  the  hands  of  an  experienced  worker,  Meier's  method  undoubtedly  yields  most 
reliable  results.  The  author,  however,  sees  no  distinct  advantage  in  these  modifications. 
He  therefore  advises  the  classical  method  for  the  beginner,  as  it  combines  simplicity 
with  correctness. 


Citron's  Standard  for  the  Strength  of  a  Reaction. 

Citron  divides  the  positive  complement  fixation  tests  into  four  grades, 
as  fellows: 


a.  Tubes  i  and  2  show  complete  absence  of  hemol- 

ysis: +  +  +  + 

b.  Tube  i  shows  complete  absence  of  hemolysis  and 

2  shows  faint  hemolysis: 


Strongly 
positive. 


CITRON'S  MODIFICATION  OF  WASSERMANN  REACTION  185 


c.  Tube  i  shows  complete  absence  of  hemolysis  and 

2  shows  complete  hemolysis:  +-f- 

d.  Tube  i  shows  partial  hemolysis  and 

2  shows  complete  hemolysis:  + 

e.  Tube  i  shows  doubtful  binding  and 


Weakly 
positive. 


2  shows  complete  hemolysis:    ' 
/.   Tubes  i  and  2  show  complete  hemolysis: — .,  Negative. 
When  a  series  of  tests  is  to  be  performed,  it  is  advisable  to  include  in  the 
reaction  three  sera  previously  tested,  one  strongly  positive,  another  weakly 
positive  and  a  third,  negative,  so  that  the  new  results  can  be  more  readily 
compared.     In  this  way  absolutely  reliable  and  constant  values  will  be 
obtained. 

Every  new  antigen  should  be  tested  for  four  weeks  before  its  practical  value  can  be 
assured.  During  this  month,  all  the  tests  should  be  done  with  both  the  old  and  new  ex- 
tract and  only  if  their  results  are  identical  should  the  new  extract  be  employed.  The 
author  is  in  the  habit  of  mixing  the  new  antigen  with  the  old  one  after  the  former  has 
proved  itself  efficient.  Occasionally  the  new  antigen  varies  in  strength  from  the  old 
one.  In  such  a  case,  if  stronger,  it  must  be  used  in  a  smaller  dose  (0.18  and  0.9)  or  if 
weaker,  must  be  used  in  larger  dose  (0.22  and  o.n).  Shaking  the  antigen  should  be 
strictly  guarded  against. 

In  order  to  control  the  effect  of  normal  liver  substances  contained  in 
the  antigen  an  extract  is  prepared  from  normal  fetal  liver  (normal  antigen). 
When  sera  from  cases  of  clinically  evident  lues  are  to  be  examined,  it  is 
unnecessary  to  have  control  tests  with  the  normal  fetal  extract  as  antigen. 
On  the  other  hand,  such  control  tests  are  absolutely  necessary  in  impor- 
tant differential  diagnosis  as  between  lues  and  carcinoma,  and  in  all 
diseases  of  the  nervous  system.  Here  a  positive  reaction  can  only  then 
be  taken  as  evidence  of  syphilis  if  the  complement  fixation  test  is  positive 
with  the  syphilitic  antigen  and  negative  with  the  normal  liver  antigen. 
If  on  the  other  hand  it  be  positive  with  both  extracts,  it  does  not  speak  for 
a  luetic  infection. 

A  strongly  positive  Wassermann  reaction  indicates  the  presence  of  a 
luetic  infection.  A  weakly  positive  result  can  be  similarly  interpreted 
if  the  serum  control  tube  (Tube  No.  3)  is  completely  hemolyzed.  If, 
however,  the  latter  still  shows  some  non-hemolyzed  red  blood  cells,  the  + 
reaction  must  be  considered  as  =*=  or  a  reaction  of  indefinite  nature. 
Only  exceptionally  are  such  doubtful  reactions  found  in  perfectly  healthy 
individuals,  although  they  are  more  often  encountered  in  different  in- 
fectious diseases  (typhoid,  measles,  scarlet  fever)  and  tumors.  A  positive 
diagnosis  of  lues  should  never  be  made  upon  a  =*=  reaction.  On  the 
other  hand  if  there  is  a  history  of  lues,  or  clinical  evidences  of  its  existence, 
a  =1=  reaction  is  to  be  interpreted  as  +  and  should  warrant  further  specific 
therapy.  As  an  end  result  of  specific  therapy  a  =*=  reaction  is  not  sufficient. 


1 86  TECHNIQUE   OF  THE   COMPLEMENT  FIXATION  METHOD 

Not  before  an  absolutely  negative  reaction  has  been  attained  should  specific 
therapy  cease. 

Several  authorities  consider  only  such  tests  as  positive  where  there  is  complete 
absence  of  hemolysis.  This  principle  is  proven  as  incorrect  by  their  own  statistics; 
a  great  number  of  their  surely  syphilitic  cases  give  a  negative  reaction. 

If  the  third  tube  (serum  control)  does  not  hemolyze,  the  test  can  be  considered 
neither  positive  nor  negative.  Very  frequently  the  third  tube  of  very  strongly  positive 
cases  will  hemolyze  very  much  more  slowly  than  negative  cases;  these  tests  must  there- 
fore remain  in  the  incubator  for  a  longer  period  than  the  negative  or  weakly  positive 
ones  and  until  the  serum  tube  is  completely  hemolyzed. 

Other  Modifications  of  Wassermann's  Technique. 

On  account  of  the  somewhat  complex  technique  of  the  reactions 
numerous  attempts  have  been  made  to  simplify  the  test  in  one  way  or 
another.  The  greatest  difficulty  lay  in  the  preparation  of  a  suitable 
antigen.  From  the  sundry  modifications  and  improvements  made  in  this 
respect,  perhaps  the  most  important  was  announced  simultaneously  by 
Landsteiner,  Mtiller  and  Potzl,  and  Forges  and  Meier. 

They  showed  that  by  alcoholic  extraction  of  luetic  and  even  normal 
organs  of  human  beings  and  lower  animals,  substances  were  obtained 
which  could  be  used  as  a  substitute  for  the  aqueous  syphilitic  antigen. 
The  belief  therefore  arose  that  the  active  agents  in  the  luetic  extract  belong 
to  the  class  of  lipoids,  and  Forges  and  Meier  endeavored  to  isolate  them 
from  the  serum.  Thereupon  it  became  evident  that  lecithin  could  replace 
the  antigen,  but  only  up  to  a  certain  point.  Further  study  by  H.  Sachs 
led  to  the  adoption  of  entire  formulae  for  artificial  antigens. 

The  new  principle  disclosed  by  these  discoveries  led  to  many  modifications  in  the 
preparation  of  the  antigen,  the  main  advantage  of  which  consisted  in  bringing  the 
reaction  into  more  general  use  and  application.  The  previous  necessity  of  making  an 
extract  from  the  liver  of  a  luetic  fetus  somewhat  limited  this.  The  Wassermann  reac- 
tion became  in  a  short  period  of  time  much  more  popular,  although  one  could  not  adhere 
to  it  with  the  same  idea  of  specificity  as  before. 

Other  changes  in  the  reaction  referred  to  the  serum  for  examination. 
H.  Sachs  demonstrated  that  the  inactivation  at  56°  C.  destroyed  a  great 
part  of  the  luetic  "reagine."  The  dispensation  of  the  latter  was  therefore 
recommended.  It  soon  became  evident,  however,  that  by  so  doing  a 
great  number  of  normal  and  non-luetic  pathological  sera  especially  from 
carcinoma  cases  gave  a  positive  reaction.  //  is  best  therefore  that  this 
modification  should  by  all  means  be  discarded. 

As  all  fresh  sera  contain  complement,  the  addition  of  guinea-pig's 
complement  seemed  superfluous  if  the  serum  for  examination  is  employed 
in  an  active  form.  The  following  combination  was  therefore  proposed: 


MODIFICATIONS    OF    WASSERMANN    TEST 


I87 


1 .  Luetic  extract  or  one  of  its  substitutes, 

2.  Active  luetic  serum  (contains  luetic  "reagine"  +  complement).     One  hour  in 
incubator. 

3.  Inactive  hemolysin. 

4.  Red  blood  cells. 

In  view  of  the  above-mentioned  objections,  especially  the  too  frequent  positive 
results,  this  modification  although  advised  by  various  authorities,  Stern  and  others, 
should  not  be  employed. 

Not  only  the  addition  of  complement,  but  also  of  immune  hemolysin 
can  be  discarded,  because  every  serum  normally  contains  hemolytic  anti- 
bodies for  foreign  species  of  blood.  The  contraindication  for  the  trans- 
fusion of  foreign  blood  depends  upon  this  principle. 

Accordingly,  some  authors  advise  the  following  schemes: 


1.  Luetic  extract  or  its  substitute. 

2.  Inactive  luetic  serum  ("luesreagine" 

-f-  hemolysin). 

3.  Complement.     One  hour  in  incubator. 

4.  Washed  erythrocytes  of  sheep. 


1.  Luetic  extract  or  its  substitute. 

2.  Active  luetic   serum   (luesreagine    -h 

complement  +  normal  hemolysin), 
one  hour  in  incubator. 

3.  Washed  erythrocytes  of  sheep. 


The  advantage  of  these  modifications  is  supposed  to  exist  in  the  omission  of  the 
immune  hemolysin.  The  preparation  and  preservation  of  this  ingredient  is,  however, 
technically  so  simple  that  this  advantage  is  only  theoretical.  Bauer  believes  that  this 
change  is  preferable  to  the  classical  method  for  the  reason  that  with  the  latter,  the  vary- 
ing amount  of  normal  hemolysin  is  always  added  to  the  constant  amount  of  immune 
hemolysin,  thereby  resulting  in  a  different  quantity  of  the  hemolysin  in  each  test.  Ex- 
perience has,  however,  shown  that  the  faint  trace  of  normal  hemoly sin  ne\er  influences 
the  result  of  the  test.  At  times  so  little  normal  hemolysin  will  exist  in  a  patient's 
serum  that  it  becomes  necessary  to  add  some  serum  of  another  normal  patient.  Such 
manipulations  lead  to  new  difficulties  so  that  taken  all  in  all,  this  innovation  offers 
no  advantages  and  should  therefore  not  be  accepted. 

Brieger  and  Renz  have  recently  advised  the  substitution  of  potassium  chlorate  for 
the  immune  hemolysin.  'Had  this  been  correct  the  biological  bases  of  the  Wassermann 
reaction  would  have  been  undermined.  Garbat  and  Munk,  however,  have  shown  that 
in  this  modification  KC103  is  entirely  inert  and  that  the  reaction  depends  upon  the 
normal  hemolysin  in  human  serum  against  sheep's  erythrocytes. 

Several  workers  in  this  field  believed  that  it  would  be  advantageous  to 
use  a  different  species  of  blood  in  place  of  sheep's  erythrocytes. 

The  only  suggestion  which  sounds  theoretically  correct  is  that  of  Noguchi,  who 
employs  human  erythrocytes  and  the  serum  of  a  rabbit  immunized  against  human 
red  blood  cells.  In  this  way  he  attempts  to  exclude  the  heterologous  normal  hemol- 
ysins,  as  human  serum  possesses  no  hemolysins  against  human  blood  cells. 


1.  Syphilis  extract  or  its  substitute. 

2.  Inactive  syphilis-serum.  ] 

3.  Complement    from    hu-      An  active 

man  being  or  guinea-      syphilis 
pig,  one  hour  in  incu-      serum, 
bator. 


1.  Syphilis  extract. 

2.  Active  defibrinated  syphilitic  blood. 

(Erythrocytes,    "reagine,"    comple- 
ment), one  hour  in  incubator. 

3.  Immune  hemolysin  of  rabbit  (injected 

with  human  blood). 


1 88  TECHNIQUE   OF   THE   COMPLEMENT  FIXATION  METHOD 

4.  Immune  hemolysin  of  a  rabbit  against 

human  erythrocytes. 

5.  Washed  human  erythrocytes.' 

From  a  practical  standpoint,  however,  no  distinct  advantage  is  offered  by  these 
modifications.  In  fact,  it  is  the  claim  of  Wassermann  and  his  pupils  that  by  the  use 
of  human  blood,  the  error  tends  toward  the  opposite  direction,  i.e.,  the  percentage  of 
positive  results  obtained  are  higher  than  is  actually  the  case. 

The  number  of  modifications  have  become  so  numerous  that  almost 
every  one  employs  his  own  "method."  There  is  absolutely  no  necessity 
for  this,  as  an  innovation  justifies  its  existence  only  if  it  is  a  distinct 
improvement,  i.e.,  discloses  a  new  fact  or  radically  simplifies  the  old. 

It  is  the  classical  Wassermann  reaction  performed  in  the  original 
manner  which  has  taught  physicians  how  valuable  a  clinical  aid  it  is. 
Their  knowledge  has  not  advanced  materially  with  all  the  new  changes. 
A  single  advantage  only  has  been  instituted  through  all  this  agitation, 
and  that  was  the  discovery  that  the  luetic  antigen  can  be  replaced  by  the 
alcoholic  extract  of  guinea-pig's  heart.  In  important  differential  diagnosis, 
however,  even  this  extract  should  not  be  considered  as  specific  as  luetic  liver 
antigen. 

For  general  work,  however,  its  employment  may  be  of  service. 

The  antigen  of  Landsteiner,  Miiller  and  Potzl  is  prepared  as  follows: 

The  heart  of  a  guinea-pig  is  washed  free  of  blood,  its  muscular  part  finely  divided  or 
macerated  in  a  mortar  and  then  extracted  with  95  per  cent,  of  alcohol  for  several 
hours  at  60°  C.  One  gram  of  the  heart  substance  should  be  mixed  with  5  c.cm.  of  the 
alcohol.  The  material  is  then  passed  through  filter-paper,  the  nitrate  being  kept  at 
room  temperature.  (The  editor  prepares  the  alcoholic  extract  by  simply  placing  the 
finely  divided  guinea-pigs'  hearts  into  95  per  cent,  alcohol  and  allowing  them  to  remain 
there  for  four  weeks  for  purposes  of  extraction.  At  the  end  of  this  period  the  alcoholic 
solution  is  titrated  and  can  be  employed  as  antigen.) 

These  authors  also  employ  the  so-called  drop  method: 

Drop        Ten  drops  of  saline  and  i  drop  of  normal  guinea-pig's  serum  as  comple- 
Method.     ment  are  placed  in  each  test-tube.     The  individual  tubes  receive  the 
following  additional  ingredients: 

First  tube:  One  drop  of  the  inactivated  serum  for  examination. 

Second  tube:  Same  as  one  +  2  drops  of  the  alcoholic  heart  extract. 

Third  tube:     One  drop  of  inactivated,  surely  luetic  serum. 

Fourth  tube:  Same  as  three  +  2  drops  of  alcoholic  heart  extract. 

Fifth  tube:  One  drop  of  inactive  normal  serum. 

Sixth  tube:  Same  as  five  +  2  drops  of  alcoholic  heart  extract. 

Seventh  tube:  Two  drops  of  extract. 

The  tubes  are  well  shaken  and  placed  in  the  incubator  for  one  hour  at 
37°  C.  Then  i  drop  of  a  .50  per  cent.  (!)  suspension  of  washed  sheep's 
erythrocytes  and  i  drop  of  hemolysin  (double  the  minimum  hemolytic 
titer)  are  added.  After  one-half  hour  in  the  incubator,  the  results  are 
read. 


MODIFICATIONS    OF   WASSERMANN   REACTION  189 

-g        ,        Bauer  entirely  excludes  the  immune  hemolysin.     His  reaction  requires 

Modifica-    the  followinS  ingredients: 

1.  Fresh  guinea-pig's  complement. 

2.  Alcoholic  organ  extract. 

3.  Five  per  cent,  sheep's  red  blood  corpuscles. 

4.  and  5.  The  inactivated  serum  for  examination  and  an  inactive  normal  control 
serum. 

Four  tubes  are  required  for  the  reaction: 

First  tube:  0.2  serum,  i.o  c.cm.  organ  extract  in  dilution  1:5  and  i  c.cm.  comple- 
ment i :  10. 

Second  tube:  Same  as  i,  but  instead  of  organ  extract,  0.85  per  cent,  sodium  chloride. 

Third  tube:  0.2  c.cm.  normal  serum,  organ  extract  and  complement  as  in  tube  i. 

Fourth  tube:  Same  as  third  tube,  but  instead  of  organ  extract  0.85  per  cent,  saline. 

The  tubes  are  placed  in  the  incubator  for  one-half  hour  and  then  i  c.cm.  of  a  5  per 
cent,  red  blood  cell  emulsion  is  added. 

After  fifteen  to  forty-five  minutes  tubes  2,  3,  and  4  show  hemolysis,  while  tube  i 
shows  hemolysis  or  not,  depending  upon  the  absence  or  presence  of  syphilis. 

Lipemic  serum  is  not  suitable  for  the  reaction, 

Bauer  asserts  that  this  method  gives  results  identical  with  those  obtained  by  the 
Wassermann  tests.  Heinrichs,  Bering  and  others  confirm  Bauer's  findings. 

If  the  alcoholic  extract  made  from  luetic  or  normal  human  or  animal  organs  is 
diluted  with  physiological  saline,  a  milky  opalescent  solution  results.  The  degree  of 
turbidity  of  the  resulting  solution  depends  upon  the  rapidity  with  which  the  saline  for 
dilution  is  added.  If  the  first  15  to  20  drops  of  the  latter  are  added  slowly,  the  resulting 
solution  will  be  much  more  turbid  than  if  the  saline  is  added  quickly.  Sachs  first 
observed  this  phenomenon  and  stated  that  the  more  marked  the  turbidity  the  more 
active  is  the  power  of  the  antigen  to  bind  complement. 

The  editor  has  worked  with  the  guinea-pig's  heart  extract  in  thousands 
of  tests  and  has  found  it  to  give  perfect  results.  The  amount  usually 
used  is  0.2  to  o.i  c.cra.  in  the  first  test-tube  and  o.i  to  0.05  in  the  second 
test-tube  as  determined  by  titration.  When  the  antigen  is  diluted  (either 
1:5  or  1:10)  the  first  c.cm.  of  saline  should  be  added  drop  by  drop  and 
shaken,  thus  producing  a  distinctly  opalescent  solution. 

The  author  refrains  from  describing  any  other  modifications  in  detail 
as  they  have  not  been  verified  sufficiently  to  merit  a  position  in  this 
important  field  of  serum  diagnosis.  This  holds  true  especially  for  the 
recently  advised  quick  and  easy  short  cuts  by  the  use  of  the  various 
ingredients  dried  on  paper.  In  order,  however,  that  one  may  acquaint 
himself  with  these  modifications,  if  he  so  desires,  the  references  to  their 
original  publications  are  here  given. 

Tschernogubow,  Berlin.  Klin.  Wochenschr.,  1908,  No.  47,  and  Deutsche 
Med.  Wochenschr.,  1909,  No.  15. 

Weidanz,  Deutsche  Med.  Wochenschr.,  1908,  No.  48,  Refer. 

Noguchi,  Journal  of  Americ.  Medic.  Associat.,  1908,  No.  22,  u. 
Munch.  Med.  Woch.,  1909,  No.  10. 

Hecht,  Wien,  Klin.  Wochenschr.,  1908,  No.  50,  and  1909,  No.  10. 


TECHNIQUE   OF   THE   COMPLEMENT  FIXATION  METHOD 

Fleming,  Lancet,  1909,  4474. 

Stern,  Zeitschr.  f.  Jmmunitatsforschung,  1909,  Bd.  I. 

Bauer,  Deutsche  Med.  Wochenschr.,  1909,  No.  10. 

IV.  Serum  Diagnosis  of  Echinococcus  Disease. 

The  technique  of  this  reaction  is  practically  the  same  as  described  for 
the  Wassermann  test. 

As  antigen  the  cystic  fluid  of  the  human  being  or  sheep  is  employed. 
The  latter  according  to  Weinberg  is  preferable,  as  human  hydatid  fluid 
sometimes  reacts  with  normal  serum. 

The  following  is  Weinberg's  outline  for  performing  the  test: 


Hydatid  fluid    Inactive 
from  sheep       from  p 

1 

serum      _ 
Complement        Hemolysm 
atient 

i 

Blood 

Results 

0.4                      o 

•  i            i 

5                       o.  i              2Xhemolytic 

i  c.cm.  5% 

o  hemolysis 

! 

dose. 

sheep's  blood. 

0.4                       o 

4                       o.i           i  2Xhemolytic 

i  c.cm.  5% 

o  hemolysis 

dose. 

sheep's  blood. 

0.4                       o 

3                       o.  i           i  2Xhemolytic 

i  c.cm.  5% 

Incomplete. 

dose. 

sheep's  blood. 

hemolysis 

0.4                       o 

2                       o.i              2Xhemolytic 

i  c.cm.  5% 

Complete. 

dose. 

sheep's  blood. 

hemolysis 

dose. 

sheep's  blood. 

hemolysis 

o  c                        o  i 

2  X  hemoly  tic 

i  c  cm.  5% 

Complete. 

04               »        01 

dose. 
2  X  hemoly  tic 

sheep's  blood, 
i  c.cm.  5% 

hemolysis 
Complete. 

o1  3                        o  i 

dose. 
2  X  hemoly  tic 

sheep's  blood, 
i  c  cm.  5  % 

hemolysis 
Complete. 

' 
02                       01 

dose. 
2  X  hemoly  tic 

sheep's  blood, 
i  c.cm.  5% 

hemolysis 
Complete. 

dose. 

sheep's  blood. 

hemolysis 

Bauer's  modification  as  employed  for  the  Wassermann  test  can  also  be 
employed  here. 


V.  Serum  Diagnosis  of  Other  Diseases. 

The  method  of  complement  fixation  can  be  employed  in  most  diseases 
where  an  antigen  can  be  made  of  the  specific  etiological  agents,  and  where 
antibodies  against  this  antigen  have  been  formed  in  the  serum  of  the 
infected  subjects.  The  relative  importance  of  the  reaction  in  a  given 


SERUM   DIAGNOSIS    OF    GONOCOCCUS    INFECTIONS  IQI 

disease  depends  upon  its  specificity  and  the  percentage  of  positive  results. 

In  epidemic  meningitis,   this  method  has  been  applied  by 

Epidemic    Bruck;   while   specific   antibodies   are   undoubtedly   formed, 

Meningitis,  the  diagnosis  can  more  readily  be  made  by  bacteriological 

examination  of  the  cerebro-spinal  fluid. 

In  tuberculosis,  Koch's  old  and  new  tuberculins  are  used 
Tuberculosis,  as  antigen.  It  is  always  best  to  use  both  of  these  prepara- 
tions (0.05-0.2)  as  occasionally  the  serum  will  fix  complement 
with  one  and  not  with  the  other  product.  The  frequency  of  spontaneously 
formed  antibodies  in  this  disease  is  not  sufficient  to  make  this  method  of 
clinical  diagnostic  importance. 

To  Miiller  and  Oppenheim  (1906)  belongs  the  credit  of  first 

Gonococcus  applying  the  complement  fixation  test  to  the  study  of  the 

Infections,   gonococcus  infections.     Meakins,  Vanned,  Wollstein,  Teague 

and    Torrey    have    all   contributed    to     this     subject,    but 

Schwartz  and  McNeil's  careful  observations  of  a  larger  number  of  cases 

have  proven  the  distinct  value  of  this  method  for  the  clinic. 

In  the  preparation  of  the  antigen,  it  is  of  paramount  importance  to  use 
as  many  different  strains  of  the  gonococcus  as  possible.  The  failure  to 
do  this  probably  accounts  for  the  many  negative  results  in  the  early 
period  of  this  test.  The  gonococci  are  best  grown  on  a  salt-free  veal 
agar,  neutral  in  reaction  to  phenolphthalein,  for  twenty-four  hours;  the 
growths  are  washed  off  with  distilled  water  and  the  emulsion  heated  in 
the  water  bath  for  two  hours  at  56°  C.  It  is  centrifugalized  and  passed 
through  a  Berkefeld  filter.  Salt  solution  is  added  to  the  antigen  just 
before  it  is  to  be  used;  it  is  then  made  up  to  0.9  per  cent,  strength,  by 
adding  one  part  of  9  per  cent,  saline  solution  to  nine  parts  of  antigen. 
The  latter  can  be  kept  indefinitely  if  preserved  in  small  quantities  in 
sealed  tubes,  heated  to  56°  C.  for  half  an  hour  on  three  successive  days. 

The  antigen  must  be  titrated  according  to  the  principles  outlined 
under  the  Wassermann  reaction.  First,  one-half  of  the  maximum  dose 
of  antigen  which  of  itself  does  not  bind  complement  is  determined;  then 
these  non-fixing  quantities  are  titrated  with  a  known  positive  human 
serum  or  a  highly  immune  rabbit  serum;  that  dose  of  antigen  is  selected 
which  binds  complement  the  strongest  with  the  smallest  amount  of  serum. 
(Parke,  Davis  &  Co.  prepares  such  an  antigen.)  The  antisheep  hemolytic 
system  is  used. 

Instead  of  using  all  the  ingredients  in  one-half  the  quantity  employed 
in  the  original  Wassermann  technique  (as  below),  one-tenth  of  the  latter 
quantity  may  be  used. 

Concerning  the  clinical  value  of  the  reaction,  Schwartz  and  McNeil 
have  thus  far  come  to  the  conclusion  that  a  positive  reaction  is  indicative 
of  a  focus  of  living  organisms,  present  in  the  body  at  the  time  or  active 


TECHNIQUE   OF   THE   COMPLEMENT  FIXATION  METHOD 
TABLE  GIVING  EXAMPLE  OF  TEST. 


Antigen 

Patient's  serum 

Complement 
10  per  cent. 

Hemolysin 

5  per  cent, 
sheep's  cells 

Hemolysis 

o.  i  c.cm. 

o.i  c.cm. 
o.  i  c.cm. 

o.  15  c.cm. 
o.  10  c.cm. 
0.05  c.cm. 

0.5  c.cm. 
0.5  c.cm. 
0.5  c.cm. 

0.5  c.cm. 
o.  5  c.cm. 
0.5  c.cm. 

0.5  c.cm. 
0.5  c.cm. 
0.5  c.cm. 

0 

o 
o 



o.  15  c.cm. 
o.i    c.cm. 

0.5  c.cm. 
0.5  c.cm. 

0.5  c.cm. 
0.5  c.cm. 

0.5  c.cm. 
0.5  c.cm. 

>    -     .        *--.  *  *  -         ";.-•• 

Complete. 
Complete. 

0.2  c.cm. 

o.  5  c.cm. 

0.5  c.cm. 

0.5  c.cm. 

Complete. 





0.5  c.cm. 

o.  5  c.cm. 

0.5  c.cm. 

Complete. 





o.  5  c.cm. 

0.5  c.cm. 

o 



0.5  c.cm. 

0.5  c.cm. 

0 

o.  i  c.cm.  of  a  posi- 
tive serum. 


0.5  c.cm. 


o.  5  c.cm.         0.5  c.cm. 


o.  i  c.cm.  of  a  nega-       o.  5  c.cm. 
tive  serum. 


0.5  c.cm.         0.5  c.crn.     !    Complete. 


only  recently.  A  negative  reaction  does  not  exclude  gonococcus  infection. 
If  the  disease  is  limited  to  the  anterior  urethra,  a  positive  reaction  is  not 
obtained;  probably  the  absorption  of  toxins  is  insufficient  to  stimulate 
the  production  of  antibodies.  The  positive  reaction  does  not  entirely 
disappear  until  seven  or  eight  weeks  after  the  patient  is  apparently  cured. 
If  it  still  persists,  one  must  be  suspicious  of  an  active  focus  somewhere. 

The  complement  fixation  test  should  be  of  special  usefulness  in  chronic 
cases  where  the  bacteriological  isolation  of  the  gonococci  is  difficult,  in 
gynecological  conditions  and  in  differential  diagnosis  of  joint  affections 
(Schwartz-McNeil,  American  Journal  of  Med.  Sciences,  May,  1911,  Sept., 
1912,  Dec.,  1912). 

That  the  complement  fixation  test  can  also  be  performed 

Typhoid    in  typhoid  fever  was  first  proven  by  Bordet  and  Gengou. 

Fever.      They  used  as  antigen  a  suspension  of  typhoid  bacilli  in  normal 

saline,  and  the  serum  from  a  convalescent  patient.  Since 
then  (1901)  numerous  contributions  referring  to  this  subject  have  been 
published,  but  the  merit  of  the  test  is  variously  interpreted.  (Widal 
and  Lesourd,  Ludke,  Leuchs,  Schone,  etc.)  The  editor  believes  that 
to  a  great  degree  the  variability  in  the  results  can  be  accounted  for 
by  the  different  antigens  employed.  Similar  to  the  findings  with  the 
gonococcus  complement  fixation  test  by  Schwartz  and  McNeil,  Garbat 
has  proven  that  a  highly  poly valent  typhoid  antigen  (made  from  numerous 


COMPLEMENT   FIXATION   TEST   IN   TYPHOID   FEVER  1 93 

strains)  is  absolutely  essential.  The  serum  from  a  typhoid  fever  patient 
gave  a  strong  complement  fixation  test  with  an  antigen  made  up  from  his 
own  typhoid  bacteria  isolated  from  the  blood  but  did  not  react  with  a 
similar  antigen  made  from  seven  other  different  strains.  The  antigen  is 
prepared  like  the  artificial  aggressins  of  Citron,  by  growing  the  bacteria  on 
agar  for  24  hours.  The  growth  is  washed  off  in  a  very  small  quantity 
of  sterile  distilled  water;  kept  at  6o°-jo°  C.  for  24  hours  (Leuchs);  after 
this  the  emulsion  is  shaken  thoroughly  with  glass  beads  for  24  hours  and 
then  centrifugalized  until  the  supernatant  fluid  is  absolutely  clear. 

The  antigen  is  titrated  as  usual  (see  Wassermann  reaction,  gonococ- 
cus  fixation  test).  The  antisheep  system  is  used. 

On  testing  the  bloods  of  thirty-six  patients  in  different  stages  of  the 
infection  with  such  an  antigen  prepared  from  twenty-eight  different 
strains,  the  editor  found  a  positive  result  in  all  but  one  instance.  [This 
patient  died  before  the  test  could  be  repeated.]  In  ten  cases  two  or  three 
examinations  were  necessary  before  the  reaction  became  positive.  For  the 
sake  of  comparison,  agglutination  tests  and  blood  cultures  were  made  at 
the  same  time.  No  distinct  relationship  between  the  three  could  be  dis- 
covered. In  several  instances  the  complement  fixation  test  appeared 
earlier  than  the  Widal  or  even  before  the  blood  culture  became  positive. 
(In  one,  confirmed  by  autopsy,  as  early  as  the  end  of  the  first  week.)  As 
a  general  rule,  however,  the  complement  fixatives  appear  later  during  the 
disease,  at  a  time  when  the  bacteria  have  disappeared  from  the  circulation. 
Thus,  this  method  becomes  of  importance  as  corroborative  of  the  Widal 
test.  The  positive  reaction  becomes  stronger  during  convalescence  and 
persists  for  several  months  after. 

The  exact  clinical  value  of  the  reaction  and  its  specificity  require 
further  statistics.  One  can  assume,  however,  that  almost  all  typhoid 
fever  patients  develop  complement  fixatives  sooner  or  later,  and  that 
these  can  be  detected  if  repeated  examinations  with  a  sufficiently  poly- 
valent antigen  are  undertaken. 

VI.  The  Differentiation  of  Proteids  by  the  Method  of  Neisser  and  Sachs. 

The  technique  employed  here  varies  only  in  a  few  details  from  the 
method  advanced  later  by  Wassermann  and  Bruck  for  the  diagnosis  of 
bacterial  infections. 

Neisser  and  Sachs  do  not  employ  a  constant  amount  of  complement  (o.i),  but  first 
titrate  the  complement  against  the  hemolysin  in  double  its  minimum  hemolytic  dose. 
For  the  test  one  and  a  half  to  two  times  the  smallest  amount  of  complement  is  necessary. 
The  hemolysin  consists  of  the  serum  of  a  rabbit  immunized  against  ox's  blood.  This 
hemolysin  acts  both  for  ox's  and  sheep's  erythrocytes. 

The  amount  of  antiserum  (for  example  antihuman  serum)  used  for  the 
test,  is  influenced  by  two  factors. 

13 


194 


TECHNIQUE   OF  THE   COMPLEMENT  FIXATION  METHOD 


1 .  An  excess  of  antiserum  can  interfere  with  the  fixation  of  complement. 

2.  The  antiserum  if  used  in  large  quantities  can  bind  complement  of  its 
own  accord,  without  the  addition  of  the  human  serum.     It  is  therefore 
best  to  ascertain  by  titration  the  smallest  quantities  of  antiserum  which 
may  satisfactorily  be  employed,  as  the  complement  fixation  test  must 
be  sufficiently  delicate  to  determine  o.oooi  c.cm.  of  the  human  serum. 

Diminishing  amounts  of  antiserum  are  mixed  with  o.oooi  c.cm.  of 
human  serum  and  o.i  c.cm.  of  complement.  A  control  series  is  made 
wherein  the  human  serum  is  replaced  by  the  same  amounts  of  saline. 
(The  quantity  in  all  tubes  should  be  made  uniform  by  the  addition  of 
normal  salt  solution,  but  the  total  amount  of  fluid  in  each  tube  should  not 
exceed  2.3  to  2.5  c.cm.)  The  tubes  are  incubated  for  i  hour  and  the 
hemolytic  amboceptor  and  red  blood  cells  added.  After  two  hours  at 
37°  C.  the  results  are  read.  The  o.oooi  c.cm.  of  the  serum  is  added  in 
the  form  of  0.2  c.cm.  of  a  i  12000  dilution. 

TABLE  III. 


Amounts  of  antiserum 

Series  A  contains  the  different 

Series  B  (control)  contains  anti- 

in  cubic  centimeters. 

quantities  antiserum+o.ooo  i  c. 

serum+o.  2    c.cm.   physiological 

cm.  human  serum  (i  :  2000.02)  + 

saline-f-o.  i  of  guinea-pig's  serum 

o.i  guinea-pig's  serum. 

One  hour  at  37°. 

One  hour  at  37°. 

+0.001  c.cm.  of  amboceptor  -f-i 

+0.001  c.cm.  of  amboceptor  +  i 

c.cm.  of  5  per  cent,  ox's  blood. 

c.cm.  5  per  cent,  of  ox's  blood. 

Hemolysis 

Hemolysis 

O.  I 

Faint  trace. 

0.075 

Faint  trace. 

0.05 

0 

0-035 

o 

•  Complete. 

0.025 

o 

0.015 

Trace. 

O.OI 

Slight. 

o 

Complete. 

The  antiserum  itself  as  seen  in  the  control  series  (B)  does  not  exhibit 
any  tendency  to  interfere  with  hemolysis,  even  in  the  amount  of  o.i  c.cm. 
(larger  quantities  never  come  into  consideration).  On  the  other  hand, 
series  (A)  shows  that  the  larger  amounts  of  the  antiserum  do  not  bind 
complement  as  thoroughly  as  the  medium  doses.  The  zone  of  complete 
complement  fixation  lies  between  0.05  and  0.025  c.cm.  of  the  antiserum. 
It  is  advisable  as  a  general  rule  to  choose  about  one  and  one-half  to  two 
times  this  minimum  quantity.  Thus  from  Table  III  it  can  be  noted  that 
0.2  c.cm.  of  a  i  :6  dilution  could  well  be  adopted  as  a  test  dose  for  com- 


DIFFERENTIATION   OF   PROTEIDS   BY   COMPLEMENT   FIXATION          1 95 

plement  fixation.  If  it  is  required  to  know  how  delicate  the  complement 
fixation  reaction  can  be  with  this  dose  of  antiserum,  the  following 
experiment  (Table  IV)  is  performed. 

Diminishing  amounts  of  human  serum  are  mixed  with  a  constant 
quantity  of  complement  and  with  this  constant  dose  of  antiserum.  At  the 
same  time  a  control  series  of  tubes  is  used,  in  which  the  antiserum  is 
substituted  by  salt  solution.  After  one  hour  of  incubation  at  37°  C. 
erythrocytes  and  hemolysin  are  added. 

TABLE  IV. 


Amounts    of    human 
serum  in  cubic  cen- 
timeters. 

Series  A  contains  human  serum 
+  i  :  6X0.2  c.cm.1  antiserum, 
+0.1  c.cm.  of  guinea-pig's  serum. 

Series  B  (control)  contains  human 
serum+o.2  c.cm.  physiological 
salt  solution+o.i  c.cm.  guinea- 
pig's  serum. 

One  hour  at  37°  . 

One  hour  at  37°. 

+0.001  c.cm.  of  amboceptor+i 
c.cm.  of  5  per  cent,  ox's  blood. 

+0.001  c.cm.  of  amboceptor+i 
c.cm.  of  5  per  cent,  ox's  blood. 

Hemolysis. 

Hemolysis. 

O.I 
O.OI 
O.OOI 
O.OOOI 
O.OOOOI 
0 

0 

o 
o 
o 
Slight. 
Complete. 

1  Complete. 

It  is  seen  from  the  above  table  that  o.ooooi  c.cm.  of  human  serum  still 
suffices  to  give  a  partial  although  incomplete  fixation  of  the  complement. 
The  delicacy  of  the  antiserum  in  this  particular  instance  is  not  very  great. 
In  forensic  practice,  the  reaction  is  carried  out  as  shown  in  Table  IV,  but 
instead  of  the  human  serum,  the  solution  of  unknown  blood  stain  in  various 
dilutions  is  titrated.  Control  series  B  should  not  be  omitted,  because  here, 
any  foreign  substance  contained  in  the  extract  and  which  might  interfere 
with  the  reaction  can  be  detected. 


i  :'6Xo.  2  c.cm.  means  o.  2  c.cm.  of  a  1:6  dilution. 


CHAPTER  XV. 
PHAGOCYTOSIS.     OPSONINS  AND  BACTERIOTROPINS. 

I.  Phagocytosis. 

By  phagocytosis  is  meant  the  taking  up,  or  engulfing  of  foreign  sub- 
stances by  certain  cells  (digesting  cells  or  phagocytes)  for  the  purposes  of 
digestion.  As  a  mode  of  nutrition,  this  is  well  known  to  exist  normally,  in 
the  lowest  unicellular  animals,  for  instance  the  amebae.  Intracellular 
digestion,  however,  can  be  traced  to  organisms  higher  in  the  scale  of  the 
animal  kingdom;  even  among  mammals  the  function  of  cell  ingestion  is 
found,  although  limited  to  a  definite  group  of  cells,  especially  those 
derived  from  the  mesoderm. 

The  inspiration  for  the  work  on  phagocytosis  and  the  greater  part  of 
its  theoretical  considerations  have  emanated  from  Metchnikoff  and  his 
numerous  pupils  at  the  Pasteur  Institute  at  Paris.  Metchnikoff  divides 
the  phagocytes  into  two  classes,  the  "  sessile  or  fixed  phagocytes,"  and  the 
"  wandering  phagocytes."  The  first  is  the  stationary  endothelial  lining  of 
blood  vessels,  and  lymph  spaces,  as  well  as  the  large  cells  of  the  spleen 
pulp  and  lymph  glands;  the  second  consists  of  the  white  blood  cells  of  the 
circulation.  From  another  standpoint  the  phagocytes  are  divided  into 
' '  microphages ' '  and  ' '  macrophages. ' '  The  former  are  practically  identical 
with  the  neutro-  and  eosinophile  polymorphonuclear  leucocytes,  while  the 
latter  present  no  distinct  group,  but  include  large  lymphocytes,  myelocytes, 
giant  cells,  etc.  The  cells  designated  as  sessile  phagocytes  also  belong  to 
the  class  of  macrophages.  The  size  of  the  cell  was  considered  by  Metch- 
nikoff as  the  deciding  feature;  not  all  macrophages  are  mononuclear  as 
generally  believed.  Thus  macrophages  appearing  in  the  peritoneal  fluid  of 
guinea-pigs  frequently  possess,  like  the  giant  cells  of  the  tubercle,  numer- 
ous nuclei.  According  to  Metchnikoff  it  is  primarily  the  microphages 
to  whom  the  function  of  bacterial  phagocytosis  is  allotted,  while  the  macro- 
phages serve  for  the  purpose  of  ingesting  dead  or  moribund  tissue  struc- 
ture. Still  there  are  certain  pathogenic  micro-organisms,  tubercle  bacilli, 
lepra  bacilli,  actinomyces,  which  are  favored  in  that  they  also  are  digested 
by  the  selective  macrophages.  The  evidence  of  phagocytosis  is  established 
by  mixing  either  in  vitro  or  vivo  the  substance  for  phagocytosis,  plus  the 
phagocytes,  and  noting  the  changes  which  ensue;  [either  in  a  stained  or 
unstained  preparation].  The  phagocytes  of  the  guinea-pig's  peritoneal 
cavity  are  especially  well  adapted  for  the  study  of  phagocytosis  in  vivo. 
The  following  experiment  of  Metchnikoff  may  serve  as  a  type. 

196 


PHAGOCYTOSIS  197 

A  guinea-pig  receives  an  intraperitoneal  injection  of  goose's  blood.  Immediately 
following  this,  the  leucocytes  disappear  from  the  peritoneal  fluid.  This  is  due  partly 
to  a  destruction  of  leucocytes  (Phagolysis)  and  partly  because  the  leucocytes  are  re- 
pulsed and  settle  upon  the  peritoneal  wall.  In  one  to  two  hours  this  so-called  negative 
phase  is  overcome  and  there  is  an  increase  of  the  leucocytes,  especially  of  the  macro- 
phages  in  the  exudate  (Hyperleucocytosis).  Now,  the  leucocytes  can  be  seen  sending 
forth  short  protoplasmic  processes — pseudopodia,  by  means  of  which  the  erythrocytes 
are  drawn  into  tHe  phagocytes.  After  a  short  time  the  macrophages  are  filled  with  the 
erythrocytes.  At  first  the  ingested  cells  appear  normal;  gradually,  however,  they  un- 
dergo changes,  which  are  clearly  visible  in  the  unstained  specimen,  indicative  of  a 
disintegrating  process,  within  the  body  of  the  phagocytes. 

."'"  .-•;•     • 

The  same  phenomenon  as  described  for  goose's  erythrocytes  can  also  be 
observed  with  bacterial  bodies. 

In  order  to  exclude  the  possible  bactericidal  influences  of  the  serum,  it  is  advisable 
when  one  is  working  with  bacteria  which  are  readily  destroyed,  as  cholera  vibrios,  to 
previously  induce  a  hyperleucocytosis  in  the  peritoneal  cavity.  The  guinea-pig  receives 
an  intraperitoneal  injection  of  10  to  20  c.cm.  of  sterile  bouillon  or  aleuronat  solution; 
in  about  twelve  hours  hyperleucocytosis  takes  place,  and  a  capillary  pipette  inserted 
into  the  peritoneal  cavity  will  withdraw  a  thick  and  turbid  exudate  of  leucocytes. 

If  this  animal  is  injected  intraperitoneally  with  bacteria,  and  a  smear  of 
the  peritoneal  fluid  made  a  short  time  after  the  inoculation,  the  bacteria 
will  be  seen  lying  within  the  microphages.  This  important  fact  has  been 
variously  interpreted.  Pfeiffer  and  his  pupils  claim  that  the  bacteria  are 
first  destroyed  or  their  virulence  greatly  diminished  by  the  bactericidal 
power  of  the  serum  and  exudate,  and  that  the  phagocytes  act  only  as 
receptacles  for  these  already  destroyed  bacteria.  Metchnikoff  believes 
that  the  phagocytes  take  up  the  living  bacteria  and  destroy  them,  thus 
representing  these  cells  as  the  most  important  weapons  of  the  organ- 
ism in  its  protection  against  infection. 

" Whenever  an  organism,  that  has  lost  its  susceptibility  to  a  par- 
ticular infection,  either  on  account  of  a  natural  born  immunity  or  one 
artificially  attained,  comes  into  conflict  with  the  etiological  agent,  a 
struggle  arises  between  the  latter  and  the  phagocytes  of  the  threatened 
individual.  The  phagocytes  appear  as  victors,  since  they  take  up  the 
bacteria  into  their  protoplasmic  bodies  and  digest  them,  thus  forever 
destroying  the  evil."  (Metchnikoff  cited  by  Levaditi.) 

Critically  considered,  there  can  be  no  doubt  that  the  phagocytes  by  their 
very  nature  are  capable  of  dealin  gwith  living  virulent  bacteria.  At  the 
same  time  one  must  observe  that  the  opsonins  and  bacteriotropins  of  the 
serum,  soon  to  be  discussed,  in  most  instances  previously  modify  the  living 
bacteria  in  a  way  at  present  still  unknown.  That,  however,  the  phagocytes 
can  ingest  bacteria  or  protozoa  which  are  alive  and  active,  has  been  demon- 
strated by  Metchnikoff 's  school.  Phagocytosis  experiments  were  under- 


1 98  PHAGOCYTOSIS.      OPSONINS  AND  BACTERIOTROPINS 

taken  with  motile  bacteria  and  spirilla.  On  microscopical  examination  it 
was  seen  that  a  phagocyte  was  in  the  act  of  taking  up  a  spirillum,  part  of 
which  was  engulfed  by  the  cell  while  the  remainder  was  still  outside  of 
the  cell  and  continuing  its  active  motility. 

Not  in  all  cases  does  phagocytosis  of  bacteria  lead  to  destruction  of  the  ingested 
microbes.  More  recent  experiments  also  seem  to  prove  that  simple  phagocytosis  of 
bacteria  must  not  be  considered  as  identical  with  their  death.  Thus,  the  exudate 
from  cases  of  anthrax  in  which  the  bacilli  lie  within  the  leucocytes,  can  still  produce 
fatal  anthrax  when  inoculated  into  animals. 

Vital  Stain-     A  more  exact  understanding  of  the  bio-chemical  nature  of 
ing  with      phagocytic  digestion  has  been  offered  by  the  method  of  vital 
Neutral  Red.  staining  with  neutral  red. 

Neutral  red  (used  as  a  i  per  cent,  solution  in  isotonic  saline)  is  a  chemical  dye  which 
stains  only  dead  cells  and  not  living  ones.  If  live  bacteria  and  phagocytes  are  mixed 
and  hanging-drop  preparations  of  these  are  made,  and  then  a  drop  of  the  stain  be  added 
to  different  preparations  at  [successively  increasing  intervals  of  time,  the  first  slide 
shows  the  extracellular  living  bacteria  unstained,  while  of  the  intracellular  bacteria,  a 
part  remains  unstained  and  the  other  colored  red. 

The  later  the  mixtures  are  stained,  the  more  numerous  are  the  intracellular  red 
stained  bacteria,  showing  that  the  ingested  micro-organisms  remain  alive  for  a  short 
time,  and  then  die.  The  intracellular  bacteria  retain  their  stain  as  long  as  the  phago- 
cytes themselves  remain  alive.  Later,  when  the  phagocytes  die,  the  formerly  red 
bacteria  lose  their  stain.  MetchnikofFs  explanation  of  the  red  staining  process  is 
that  during  the  act  of  digestion  by  the  phagocytes  an  acid  ferment  is  liberated  which 
gives  the  color  reaction  with  the  neutral  red. 

For  many  years  MetchnikofFs  phagocytic  theory  opposed  the  concep- 
tion of  Ehrlich  and  also  Pfeiffer  in  relation  to  the  importance  of  ambo- 
ceptor  and  complement  in  the  mechanism  of  immunity.  It  would  be  out 
of  place  here  to  review  the  various  experiments  performed  and  offered  on 
each  side  in  explanation  of  its  standpoint.  Suffice  it  to  say  that  Metch- 
nikoff  denied  the  existence  of  free  complement  within  the  animal  organism. 
He  moreover  claimed  that  the  complement  was  found  normally  only  in 
the  phagocytes  and  hence  called  it  "cytase,"  differentiating  the  two 
phagocyte  groups  as  "micro-  and  macrocytase."  The  "cytase"  is 
liberated  when  the  phagocytes  are  broken  up.  The  amboceptors  are 
considered  as  split  products  of  the  phagocytes  and  known  by  Metchni- 
koff  as  "fixators." 

2.  Opsonins. 

In  recent  years  the  closer  agreement  which  has  arisen  between  the 
followers  of  phagocytic  and  humoral  theories  was  made  possible  by  the 
fact  that  Denys  and  Leclef,  Leishmann,  Wright  and  Douglas  and  others, 


PRINCIPLES    OF   THE    OPSONIC   INDEX  199 

demonstrated  that  phagocytosis  occurs  in  most  cases  only  in  the  presence 
of  serum.  If  the  phagocytes  are  thoroughly  washed,  so  that  they  are 
entirely  serum-free,  phagocytosis  will  not  take  place,  or  will  do  so  imper- 
fectly. The  belief  of  some  authors  that  " spontaneous  phagocytosis" 
without  serum  was  altogether  impossible  was  disproved,  especially  by 
Lohlein.  The  manner  in  which  the  serum  acts,  whether  it  stimulates  the 
digestive  activity  of  the  leucocytes  or  whether  it  so  changes  the  bacteria 
that  they  can  more  readily  be  taken  up  by  the  phagocytes,  has  been  settled 
in  favor  of  the  latter  view  through  researches,  especially  of  Wright  and  his 
followers  as  well  as  of  Neufeld.  The  substances  within  the  serum  which 
thus  modify  the  bacteria  have  been  designated  by  Wright  as  "opsonins." 
C'opsono"  =  I  prepare  food  for.) 

Opsonins  are  demonstrated  by  mixing  bacteria,  serum  and  washed 
leucocytes,  allowing  this  mixture  to  remain  in  the  incubator  for  a  short 
time,  and  then  staining  smear  preparations.  Wright  then  counts  a 
certain  number  of  leucocytes  and  the  number  of  bacteria  found  within 
these  leucocytes.  The  relation  between  the  number  of  ingested  bacteria 
and  the  counted  number  of  phagocytes  is  designated  as  the 
The  Opsonic  phagocytic  count.  Wright  compared  the  phagocytic  counts 

Index.  of  infected  individuals  with  those  of  normal  persons  and  found 
that  those  of  the  former  were  much  lower.  The  relation  existant 
between  the  two  he  expressed  in  the  form  of  a  fraction  and  that  is  known  as 
the  opsonic  index.  Thus  a  smear  made  from  a  mixture  of  equal  parts  of 
an  emulsion  of  staphylococci,  leucocytes  and  the  patient's  serum  showed 
for  example  75  cocci  to  100  leucocytes;  while  one  made  from  a  mixture 
of  equal  parts  of  the  same  bacterial  emulsion  and  leucocytes,  but  a 
normal  individual's  serum,  demonstrated  150  bacteria  to  100  leucocytes. 
The  opsonic  index  of  the  patient's  serum  was  therefore  one-half  (0.5). 

According  to  Wright,  the  opsonic  index  expresses  the  animal's  resist- 
ance to  infection.  He  believes  that  a  low  opsonic  index  for  a  given 
bacterium  indicates  a  susceptibility  on  the  part  of  the  individual  for  that 
particular  infective  agent.  Furthermore,  the  opsonic  index  he  claims 
can  be  used  as  an  aid  in  the  diagnosis  of  infectious  diseases,  inasmuch  as 
opsonins  are  specific.  Thus  the  opsonic  index  in  a  tuberculous  individual 
is  low  only  for  the  tubercle  bacillus  and  not  for  other  bacteria. 

When  an  animal  is  immunized,  its  opsonic  index  toward  the  respective 
bacterium  is  considerably  increased.  The  question  has  been  asked 
whether  the  immune  opsonins  formed  during  this  process  are  identical 
with  the  normal  opsonins.  Wright  and  a  number  of  the  more  recent 
authorities  believe  that  they  are  different.  Neufeld,  who  discovered  these 
immune  opsonins  independently  of  Wright,  named  them  Bacteriotropins, 
and  pointed  out  that  while  the  normal  opsonins  are  destroyed  when  heated 
to  56°,  the  bacteriotropins  remain  unharmed.  As  yet  the  exact  nature 


200 


PHAGOCYTOSIS.       OPSONINS   AND  BACTERIOTROPINS 


of  the  immune  as  well  as  of  the  normal  opsonins  has  not  been  clearly 
defined.  It  is  still  a  matter  for  investigation  whether  in  the  case  of 
opsonins  one  is  dealing  with  entirely  new  substances  or  whether  they 
are  the  old  well-known  bodies  like  the  agglutinins,  complements  and 
amboceptors  with  a  new  action. 

The  fact  that  the  opsonic  index  is  raised  by  immunization  while  it  is 

usually  found  diminished  during  spontaneous  infection    in    man,   led 

Wright  to  believe  that  good  results  may  be  obtained  by 

Increase  of  increasing  the  opsonic  index  of  the  already  infected  individual 

Opsonic     by  means  of  immunization.     In  this  way  he  thought  the 

Index  by  Im- patient's   predisposition   to    the   particular   infection   would 

munization.  be  overcome.     Wright's  experiments  showed  that  the  opsonic 

index  could  be  increased  by  injection  of  extremely  small 

doses  of  dead  bacteria  (Wright's  vaccines.) 

If  an  individual  suffering  from  an  acne  or  furunculosis,  and  who  has  a  low  opsonic 
index  for  the  staphylococcus,  is  injected  with  a  very  small  number  of  staphylococci,  his 
opsonic  index  sinks  still  more  for  a  short  period  after  the  inoculation  (negative  phase). 

This  is  explained  by  the  fact 
that  the  injected  bacteria  absorb 
the  existing  opsonins.  New 
opsonins  are,  however,  then 
produced,  which  immediately 
make  up  for  the  loss  occasioned 
during  the  negative  phase,  with 
the  result  that  after  several  days 
there  is  an  increase  of  the  opsonic 
index  (positive  phase)  which 
lasts  for  a  short  time.  Then 
the  index  again  begins  to  fall,  as 

the   stimulus   to  the   formation 
CHART  5. — Curve  of  the  opsonic  index  following  the     £  .  . 

inoculation  of  a  small  dose  of  staphylococcus  vaccine.  of  °Psonms  1S  transitory.  It 
The  arrow  indicates  the  time  of  injection.  usually  sinks  to  below  the  nor~ 

mal  level,  only  to  rise  again  to  a 

point  slightly  above  the  normal,  where  it  remains  stationary.  This  irregular  curve 
represents  the  typical  course  of  the  opsonic  content  of  the  blood  after  a  vaccine  injec- 
tion; apart  from  this  characteristic  picture  numerous  exceptions  exist.  Thus  by  the 
use  of  very  minute  bacterial  doses,  the  negative  phase  immediately  following  the 
injection  is  entirely  absent.  Reversely  very  large  doses  exhibit  a  prolonged  nega- 
tive phase. 

Wright  graphically  represents  these  variations  in  the  opsonic  index  by 
charts,  an  example  of  which  is  given  here  (Chart  5). 

In  order  that  the  therapeutic  effect  may  persist,  it  is  advisable  to 
repeat  the  inoculation.  A  new  injection  should  be  given  at  the  height 
of,  or  during  the  positive  phase,  as  an  inoculation  repeated  during  a 
negative  phase  will  result  in  further  depression  of  the  index  to  a  very 
low  level.  It  is  even  possible  in  this  way  to  harm  the  patient.  The 


Opsonic 
Index 


Normal 


A 

\ 

i 

\ 

i 

, 

t 

/ 

\ 

/ 

^ 

s  \ 

^ 

/ 

\ 

/ 

\ 

f 

V 

V 

9 

10 

»J1 

12 

13 

m 

15 

16 

17 

18 

19 

20 

21 

22 

AUTO-INOCULATION 


201 


poor  results  obtained  during  the  first  era  of  tuberculin  treatment  can, 
according  to  Wright,  be  attributed  to  the  failure  of  this  observation.  It 
is  the  production  of  cumulative  positive  phases  that  is  the  aim  of  vaccine 
treatment.  (Chart  6.) 

Wright  and  his  co-workers  have  noticed  that  an  increase  in  the  opsonic 
index  usually  runs  parallel  with  an  improvement  in  the  condition  of  the 
patient. 

Inasmuch  as  an  increase  in  the  opsonic  index  is  occasioned  by  introduc- 
ing into  the  general  system  even  a  very  small  number  of  bacteria,  it  seems 
probable  that  such  spontaneous  inoculation  will  take  place  during  the 
course  of  an  infectious  disease.  In  fact,  a  spontaneous  rise  in  the  opsonic 
index  is  observed  during  convalescence  or  after  the  crisis  of  an  infection. 
A  high  index  is,  however,  also  noticed  at  other  times;  for  example,  tuber- 

Nov.      Dec. 
30  1  Z  3  4 


12  13 14  1516 17 18  192021  222324252627282930 


CHART  6. — Opsonic  curve  during  treatment  with  New  Tuberculin. 

culous  individuals  show  a  higher  index  than  normal  persons.  Wright 
explains  this  by  the  so-called  " auto-inoculation;"  for  example,  after 
moderate  exercise,  or  work,  tuberculin  is  liberated  from  the  tuberculous 
focus  and  in  this  way  acts  like  a  therapeutic  injection  of  tuberculin,  i.e., 
the  index  will  be  raised.  Therefore,  an  excessively  high  opsonic  index  is 
of  just  as  great  diagnostic  value  as  a  low  one.  Wright  furthermore 
believes  that  constant  irregularities  or  variations  in  the  height  of  the  opsonic 
curve  serve  as  plausible  evidence  for  the  existence  of  infection,  because 
under  normal  circumstances  the  curve  should  remain  at  a  level.  Not 
infrequently,  however,  cases  come  under  observation  where  in  spite  of  a 


202 


PHAGOCYTOSIS.      OPSON1NS  AND  BACTERIOTROPINS 


Dpsonii: 
Index. 


2.4 
2.2 
2.0 

16 

14 
7,2 

Normal    1 0 

as 

0,6 


Gono-Opsonic   Index.      •    •  -•- 


Tuberculo-Opsontc  Index 


MINIMUM 


CHART  7. — Increase  in  the  opsonic  index  for  the  gonococcus  brought  about  by  Bier's 

hyperemia. 


Feb. 


CHART  8. — Tuberculin   auto-inoculation   following  physical  examination   and   massage. 

(Tuberculous  Lymphadenitis.) 


AUTO-INOCULATION  203 

distinct  evidence  of  the  existence  of  an  infection  the  opsonic  index  remains 
normal.  In  such  instances,  for  some  reason,  the  bacteria  and  their  products 
do  not  reach  the  general  circulation  and  therefore  no  occasion  is  offered 
for  either  an  elevation  or  sinking  of  the  opsonic  index.  Wright  and 
Freeman  were  able  to  show  that  all  active  and  passive  motions  of  an  infected 
joint,  as  well  as  any  vascular  changes  which  induce  a  flow  of  lymph  toward 
the  focus  of  infection,  lead  to  auto-inoculations,  which  are  manifested  in  a 
change  of  the  opsonic  index.  Such  artificial  production  of  auto-inocula- 
tion can  be  employed  in  various  forms  as  a  means  of  diagnosis:  thus,  in 
articular  rheumatism,  massage;  in  pulmonary  tuberculosis,  breathing 
exercises;  in  laryngeal  diseases,  loud  reading;  and  in  tuberculosis  of  the 
lower  extremities,  active  gymnastics  will  occasion  changes  in  the  opsonic 
curve. 

An  example  is  given  in  Chart  7.  The  patient  was  a  woman  with  a  swollen  wrist 
joint.  In  order  to  decide  whether  this  was  a  gonorrheal' or  tuberculous  process,  the 
opsonic  index  was  taken  and  found  to  be  0.94  to  0.97  for  the  gonococcus  and  1.03  to 
1.35  for  the  tubercle  bacillus.  As  these  figures  differed  very  slightly  from  the  normal, 
the  test  was  repeated,  but  this  time  after  Bier's  hyperemia  had  been  applied  and  the 
forearm  placed  into  warm  water  for  one  hour.  The  opsonic  index  for  the  tubercle 
bacillus  remained  the  same,  while  that  for  the  gonococcus  had  undergone  marked 
variations. 

A  similar  experiment  with  a  woman  having  tuberculous  lymphadenitis  is  given  in 
Chart  8. 

Wright  makes  use  of  these  variations  of  index  caused  by  auto-inocula- 
tion in  determining  the  prognosis  of  a  case.  An  infection  is  only  then 
considered  cured  when  artificial  auto-inoculation  is  no  longer  possible. 

The  Technique  for  the  Determination  of  the  Opsonic  Index. 

For  the  determination  of  the  opsonic  index  are  required, 

1.  Serum  of  the  patient. 

2.  Serum  of  the  normal  individual  (as  control). 

3.  Washed  blood  cells  (Leucocytes). 

4.  Bacterial  emulsion. 

The  blood  serum  is  obtained  from  the  finger  tip  at  the  root  of  the  nail. 
It  is  most  efficacious  to  first  produce  a  hyperemia  of  this  part  by  constrict- 
ing the  finger  either  with  a  narrow  gauze  bandage  or  a  small  soft  rubber 
tube  (the  editor  has  found  the  latter  much  more  convenient) .  The  prick  is 
then  made  with  a  needle  or  finely  drawn  out  glass  tube.  The  blood  flows 
spontaneously  and  is  collected  into  one  of  Wright's  capillary  tubes  (Fig. 
19)  by  approximating  the  curved  end  of  the  latter  to  the  blood  (Fig.  18). 
The  straight  capillary  end  of  the  tube  (away  from  the  blood)  is  then 
warmed  in  a  small  flame  and  sealed.  The  tube  is  laid  down  flat,  and 


204 


PHAGOCYTOSIS.      OPSONINS   AND  BACTERIOTROPINS 


allowed  to  cool;  by  the  cooling  the  blood  is  sucked  back  from  the 
unsealed  capillary  end;  this  end  may  also  be  sealed  in  the  tip  of  the 
flame.  The  blood  then  coagulates  and  the  serum  separates  off.  The 
separation  of  the  latter  may  be  hastened  by  centrifugalization  for  a 
short  time. 


FIG.  i 8. 


FIG.  19. 


FIG.  20. 


In  order  to  obtain  leucocytes,  a  small  test-tube  which  holds  3  to  4  c.cm. 
is  filled  2/3  with  a  1.5  per  cent,  solution  of  sodium  citrate,  and  about  6 
to  7  drops  of  blood  from  a  healthy  individual"  are  collected  in  .this 
solution  (Fig.  21).  The  tube  is  inverted  several  times  to  thoroughly  m 


FIG.  21. 


FIG.  22. 


the  blood  so  that  the  citrate,  by  precipitating  the  calcium  salts  of  the 
blood,  effectively  prevents  coagulation.  The  suspension  is  centrifugalized 
until  the  corpuscles  are  thrown  down  and  a  distinct  white  layer  (leucocytes) 
is  seen  upon  the  surface  of  the  red  cells  (Fig.  22).  The  clear  supernatant 
citrate  solution  is  pipetted  off,  care  being  taken  not  to  disturb  the  white 
layer.  Some  0.85  per  cent,  saline  is  added,  mixed  and  the  mixture  again 


PREPARATION   OF   BACTERIAL   EMULSION 


205 


centrif  ugalized.  The  washing  with  normal  saline  solution  is  repeated  once 
or  twice  and  as  much  of  the  clear  liquid  as  possible  is  finally  removed;  the 
remaining  cells  are  thoroughly  mixed  and  in  this  form  are  ready  for  use. 
The  bacterial  emulsions  with  the  exception  of  the  tubercle  bacillus  are 
made  from  agar  cultures ;  the  growths  of  gram  +  cocci  may  be  as  old  as 
twenty-four  hours,  while  the  coliform  organisms  and 
the  gram  —  cocci  are  preferably  used  only  four  to  ten 
hours  old  (the  younger  the  better).  A  loopful  of  cul- 
ture from  an  agar  tube  is  thoroughly  rubbed  up  with 
several  drops  of  salt  solution  in  a  watch-glass  by  means 
of  a  small  glass  pestle.  The  salt  solution  is  best  added 
very  gradually,  drop  by  drop,  in  order  to  make  a  more 
perfect  emulsion.  This  may  then  be  advantageously 
centrifugalized  for  a  short  period,  to  bring  down  the 
large  clumps.  The  supernatant  opalescent  portion  is 


Mar 


FIG.  24. 


taken  off  for  use,  thoroughly  mixed,  and  if  necessary  diluted.  Emul- 
sions of  coliform  organisms  are  the  most  easily  made.  Frequently  it  is 
sufficient  to  rub  up  with  the  platinum  loop  a  loopful  of  such  bacteria 
on  the  side  of  a  small  test-tube  containing  saline.  The  proper  thickness 
of  the  resulting  emulsion  varies.  As  a  rule,  bacillary  emulsions  are  re- 


206  PHAGOCYTOSIS.      OPSONINS  AND  BACTERIOTROPINS 

quired  to  have  a  thicker  appearance  to  the  naked  eye  than  coccal  ones. 
The  latter  should  be  only  slightly  opalescent. 

In  order  to  make  a  satisfactory  tubercle  emulsion,  a  more  elaborate 
method  is  necessary.  The  dead  and  dried  tubercle  bacilli  are  employed 
for  this  purpose.  A  portion  of  these  bacilli  is  very  thoroughly  triturated 
in  an  agate  mortar,  or  between  two  slides,  or  in  a  grinder  devised  for  this 
purpose,  at  first  alone  and  then  with  1.5  per  cent,  salt  solution  added 
drop  by  drop.  In  this  way  a  paste,  and  subsequently  a  comparatively 
thick  emulsion  is  made.  For  use,  a  small  portion  of  the  resultant  emul- 
sion is  centrifugalized  until  the  upper  layers  are  fairly  opalescent. 

These  upper  layers  are  pipetted  off,  and  thoroughly  mixed.  A  smear 
of  this  should  be  made  and  stained  in  order  to  observe  that  the  emulsion 


FIG.  25. 

is  free  from  clumps  and  not  too  thick.  Such  an  emulsion  sealed  up  in  a 
glass  tube  and  sterilized  at  60°  C.  for  i  hour  can  be  kept  for  about  one 
week. 

Streptococci  may  similarly  be  rubbed  up  in  a  mortar  with  0.85  per 
cent,  salt  solution  and  then  centrifugalized.  As  a  rule,  however,  vigorous 
pipetting  into  a  watch  glass  with  subsequent  centrifugalization  for  a  few 
minutes  is  sufficient  to  break  up  the  chains  and  leave  a  satisfactory 
emulsion. 

If  several  specimens  of  blood  are  to  be  examined  it  is  best  to  do  a 
preliminary  phagocytic  count  in  order  to  test  the  strength  and  condition 
as  regards  clumping  of  the  emulsion.  The  phagocytic  count  for  tubercle 
should  be  between  1.5  to  2  per  cell  and  for  other  organisms  not  less  than 
3  per  cell.  According  to  the  preliminary  finding  further  dilution  or 
concentration  of  the  emulsion  is  necessitated.  The  pipe  tes  employed 
for  the  opsonic  index  should  b  about  16  cm.  long  and  made  from  glass 
tubing  about  5/16  of  an  inch  in  diameter.  They  should  all  be  approxi- 


PREPARATION   OF    SMEAR 


207 


mately  of  the  same  caliber  and  but  slightly  tapering  toward  the  point. 
The  rubber  nipple  should  tightly  fit  the  piece  of  tubing  or  bulb  available. 
For  use,  the  capillary  end  should  be  cut  square  and  the  pipettes  marked 
with  a  paraffin  pencil  about  3/4  of  an  inch  from  their  extremity.  The 
content  as  far  as  this  mark  is  the  unit  of  volume  in  each  case. 

The  rubber  nipple  is  now  held  between  thumb  and  forefinger  and 
gently  compressed,  the  capillary  end  introduced  into  the  well-mixed 
blood  cells  and  the  unit  volume  drawn  up  by  slightly  relaxing  the  pressure 
on  the  bulb.  Next  a  tiny  bubble  is  allowed  to  enter,  then  an  equal  volume 
of  the  emulsion,  followed  by  another  tiny  bubble  which  latter  is  succeeded 
by  an  equal  volume  of  serum.  By  gentle  pressure  on  the  bulb  the  several 
volumes  are  ejected  upon  a  clean  glass  slide,  and  thoroughly  mixed  by 
alternately  sucking  the  mixture  into  the  pipette  and  squeezing  it  out  again 
upon  the  slide.  It  is  enough  to  repeat  this  action  three  times.  Then  the 


FIG.  26. 

mixture  is  drawn  up  into  the  pipette,  the  end  sealed  in  a  small  pilot  flame, 
the  pipette  placed  into  the  opsonizer  (Fig.  24)  and  the  time  noted.  This 
operation  is  repeated  with  each  serum. 

Coliform  organisms  and"  the  gram  negative  cocci  should  be  incubated 
not  longer  than  six  to  eight  minutes.  Tubercle  bacilli  and  other  organisms 
require  fifteen  minutes,  more  or  less  according  to  the  strength  of  the 
emulsion. 

The  pipettes  are  withdrawn  in  the  same  order  in  which  they  were 
placed  in  the  opsonizer.  The  contents  of  each  are  blown  out  on  to  a 
slide,  and  very  carefully  mixed  as  before  (Fig.  25).  The  entire  quantity 
is  divided  between  two  or  three  slides  and  several  smears  are  made,  the 
best  one  being  selected  for  counting.  These  slides  should  previously  have 
been  roughened  with  very  fine  (oo)  emery  paper  and  cleaned  with  a  duster, 
and  should  rest  on  their  concave  surface  so  that  the  smear  is  made  on  the 
convex  side.  (It  will  be  noticed  that  a  slide  can  be  made  to  rotate  if 
resting  on  one  surface  (convex),  but  does  not  do  so  when  resting  on 
the  concave  surface.)  The  smears  are  best  made  by  means  of  a 
broken  slide  with  a  slightly  concave  edge.  This  "spreader"  (Fig.  26)  is 
made  by  sharply  breaking  a  glass  slide  at  about  its  middle,  this  being 


208 


PHAGOCYTOSIS.      OPSONINS  AND  BACTERIOTROPINS 


facilitated  by  scratching  the  edges  of  the  slide  with  a  glass  cutter  at  the 
point  where  it  is  desired  to  break  it.  The  editor  has  broken  as  many  as 
twenty  to  thirty  slides  before  a  proper  spreader  was  obtained.  It  pays 
to  do  this,  because  upon  the  sharpness  of  the  fracture  and  cleanliness  of 
the  spreader  depends  the  edge  of  the  film,  and  secondarily  the  ease, 
rapidity,  and  accuracy  of  the  count.  If  the  film  be  well  made,  it  will 
have  a  straight  edge  within  which  will  be  found  practically  all  the  leuco- 
cytes, as  they  are  larger  than  the  red  blood  cells,  and  therefore  dragged 
to  the  end  of  the  film. 


FIG.  27. — Phagocytosis  of  tubercle  bacilli. 

The  preparations  are  fixed  in  a  saturated  solution  of  corrosive  subli- 
mate for  two  or  three  minutes,  washed  with  water,  and  stained  with 
methylene  blue  or  carbol-thionin  (1/4  per  cent,  thinonin,  and  i  per  cent, 
carbolic  acid).  Carbol  thionin  is  preferable.  It  should  be  slightly  diluted 
and  warmed  before  being  poured  upon  the  slide.  Here  it  is  allowed  to 
remain  for  several  minutes,  then  washed  off  in  water,  and  the  slide  dried 
with  filter-paper.  The  tubercle  films  are  best  fixed  with  formalin  vapor, 
stained  with  hot  carbol  or  aniline  fuchsin,  decolorized  in  2.5  per  cent,  of 
H2SO4,  treated  with  4  per  cent,  acetic  acid  to  dissolve  the  erythrocytes  and 
counter  stained  with  1/2  per  cent,  of  methylene  blue  in  1/2  per  cent,  of 
sodium  carbonate.  It  is  most  important  that  tubercle  films  be  carefully 
stained  because  it  is  desirable  to  color  every  bacillus  and  yet  not  break  up 
the  leucocytes  (Fig.  27). 

With  a  i/i 2  inch  oil  immersion  lens  a  minimum  number  of  one  hundred 


DETERMINATION    OF    OPSONIC   INDEX  2OQ 

polymorphonuclear  leucocytes  are  now  examined  and  the  number  of 
microbes  they  contain  enumerated. 

Similar  calculation  is  undertaken  with  the  normal  control  serum.  The 
fraction  obtained  by  dividing  the  number  of  bacteria  contained  in  100  cells 
on  the  patient's  slide,  by  the  number  in  100  cells  on  the  normal  slide,  gives 
the  opsonic  index  of  the  patient's  serum. 

For  example,  the  normal  individual  has  284  and  the  patient  262  bacteria 
in  100  cells,  the  fraction  which  gives  the  patient's  opsonic  index  would  be 
262/284  °r  0.92. 

The  principle  of  Wright's  technique  is  simple,  but  it  requires  a  great 
deal  of  practice  before  it  is  mastered.  Only  then  are  the  results  reliable. 
One  must  adopt  the  same  principles  when  counting  the  control  slide  as 
when  the  patient's  film  is  counted.  If  in  the  last  case,  for  instance,  the 
cocci  situated  on  the  edges  of  the  cells  are  not  included  in  the  count, 
they  should  also  be  excluded  in  the  first  case.  The  absolute  count  is  of  no 
importance.  It  is  the  relative  proportion  which  is  significant. 

As  a  normal  control,  it  is  best  to  take  the  average  of  the  phagocytic 
counts  of  a  series  (3  to  4)  of  normal  sera  or  first  equally  mix  the  different 
sera,  and  take  the  phagocytic  count  of  the  pool. 

Normal  sera  should  not  differ  from  one  another  in  a  tubercular  opsonic 
estimation  by  more  than  10  per  cent. 

Wright's  Vaccine  Treatment. 

As  has  been  said,  the  principle  of  Wright's  vaccine  treatment  depends 
upon  the  immunization  with  small  doses  of  dead  bacteria,  so-called  vac- 
cines, whereby  the  opsonic  index  of  the  individual  is  raised.  This  is  usu- 
ally associated  clinically,  with  improvement  in  the  patient's  condition. 

The  effect  of  the  immunization  according  to  Wright  depends  upon: 

1.  Individual  reaction  of  the  patient. 

2.  Preparation  of  the  vaccine. 

3.  Dosage  and  form  of  application. 

The  individual  reaction  of  the  patient  can  be  measured  by  the  opsonic 
index. 

As  far  as  the  preparation  of  the  vaccine  is  concerned,  Pasteur's  conten- 
tion that  a  vaccine  must  necessarily  be  made  up  of  living  cultures  has 
not  proved  itself  correct.  Carefully  killed  cultures  suffice  in  almost  all 
cases.  An  example  of  the  preparation  of  Wright's  vaccine  is  here  given. 

The  Preparation  of  a  Staphylococcus  Vaccine. 

Agar  cultures  are  grown  for  twenty-four  hours,  and  about  3  c.cm.  of 
sterile  normal  saline  solution  are  added  to  each  culture.  The  growth  is 
washed  off  in  the  saline  solution  by  means  of  a  platinum  needle  or  freshly 

14 


210  PHAGOCYTOSIS.      OPSONINS  AND  BACTERIOTROPINS 

prepared  capillary  pipette.  The  suspension  of  bacteria  is  placed  in  a 
sterile  test-tube,  the  end  of  this  tube  drawn  out  in  the  blow-pipe  flame  and 
sealed.  The  drawn-out  portion  should  be  about  2  inches  in  length  and  as 
strong  as  possible.  The  emulsion  is  now  vigorously  shaken  for  fifteen 
minutes.  The  extremity  of  the  drawn-out  tube  is  then  cut  and  a  few 
drops  of  the  emulsion  expelled  into  a  clean  watch  glass,  or  a  small  part  of 
the  drawn  end  is  cut  off  so  that  a  portion  of  the  emulsion  is  still  contained 
within  it.  The  tube  is  resealed,  and  then  submerged  in  water  kept  at  60° 
C.  for  one  hour.  This  usually  suffices  to  kill  the  bacteria. 

The  small  amount  placed  in  the  watch  glass  or  in  the  capillary  test- 
tube  serves  for  the  standardization,  which  is  carried  out  as  follows:  A 
pipette  with  rubber  bulb,  as  prepared  for  the  opsonic-index  test,  is  also  used 
here.  A  volume  of  freshly  drawn  blood  of  known  corpuscular  content, 
best  taken  from  the  worker's  own  finger,  and  an  equal  volume  of 
bacterial  emulsion  are  mixed  thoroughly  with  six  or  seven  volumes  of  i  1/2 
per  cent,  citrate  solution;  several  even  films  (which  may  be  fairly  thick), 
are  then  made  by  means  of  the  ordinary  edge  of  a  slide,  and  stained  with 
carbol-thionin,  Leishmann's  or  Jenner's  stain. 

The  entire  smear  is  divided  up  (with  a  blue  skin  pencil)  into  eight 
equal  subdivisions,  by  one  transverse  line  drawn  parallel  to  the  long  diam- 
eter of  the  slide  at  its  middle  and  five  vertical  lines,  one  at  each  edge  of  the 
smear,  one  in  the  center  and  one  equally  distant  between  the  edge  and  the 
central  line.  It  is  also  advantageous  to  employ  an  eye  piece,  the  field  of 
which  has  been  divided  or  made  very  much  smaller  by  the  insertion  of  a 
paper  screen  with  a  small  central  opening  representing  the  size  of  the 
desired  field.  Five  or  six  fields  are  then  counted  in  each  of  the  eight  sub- 
divided areas.  The  number  of  red  blood  cells  seen  in  each  field  are  enumer- 
ated in  one  vertical  column,  the  number  of  organisms  in  the  same  field  in 
another  column.  In  this  manner  an  average  of  the  entire  slide  is  obtained. 

By  means  of  a  simple  proportional  sum,  the  number  of  bacteria  per 
cubic  centimeter  of  emulsion  is  estimated,  e.g.,  the  number  of  red  blood  cells 
counted  is  850  and  the  number  of  bacteria  1020.  The  red  blood  corpuscles 
used  in  the  standardization  are  known  to  number  5,000,000  to  a  cubic 
millimeter  or  5,000  million  to  a  cubic  centimeter;  therefore  the  number  of 
bacteria  to  a  cubic  centimeter  of  the  unknown  emulsion  is  expressed  as 
follows. 

850  : 1020  :  :  5,000,000,000  :  No.  of  bacteria  per  c.cm.  of  emulsion, 

.*.  6,000,000,000  =  the  number  of  bacteria  per  c.cm.  of  emulsion. 

After  the  emulsion  has  been  heated  for  one  hour,  the  tube  is  opened 
and  a  drop  is  expressed  into  an  agar  culture  tube  which  is  incubated  for 
twenty-four  hours  to  demonstrate  whether  the  emulsion  is  sterile  or  not. 
At  the  end  of  this  time,  if  a  growth  is  observed,  the  emulsion  must  be  heated 
again  for  one  hour  at  60°  C/and  its  sterility  again  tested  for. 


CONCENTRATIONS    OF   DIFFERENT  VACCINES  211 

Proper  dilution  of  the  emulsion  is  next  undertaken.  Small  bottles 
containing  25  c.cm.  of  1/2  per  cent,  carbolic  acid  in  sterile  saline  are  aseptic- 
ally  closed  with  rubber  caps;  for  example,  it  is  desirable  to  make  up  these 
25  c.cm.  with  staphylococcus  vaccine  so  that  each  cubic  centimeter  contains 
500  million  bacteria,  then 

(desired  amt.  to  each  c.cm.) 
500,000,000  X   5  No.  of  c.cm.  desired 


6,000,000,000  (dose  of  original  emulsion  per  c.cm.) 

2.08  c.cm;  or  approximately  2  c.cm.  of  the  original  emulsion  must  be  added 
to  the  25  c.cm.  (to  be  exact  23  c.cm.)  to  make  up  the  desired  dilution. 

The  rubber  cap  is  finally  coated  with  melted  paraffin  wax. 

For  stock  vaccines  it  is  best  to  make  up  the  different  vaccines  in  the  fol- 
lowing concentrations: 

1.  Staphylococcus  vaccine — prepared  from  various  strains  of  staphy- 
lococcus, aureus,  citreus,  and  albus,  in  three  concentrations:  1000  million, 
500  million  and  100  million,  to  the  c.cm. 

2.  Streptococcus  vaccine  in  20  mil.,  10  mil.  and  5  mil.  concentrations. 
Since  the  streptococcus  grows  very  sparingly,  cultures  of  two  or  three  days' 
growth  may  have  to  be  employed  for  the  preparation  of  a  vaccine,  and  even 
then  it  may  be  necessary  to  use  one  broth  culture  instead  of  sterile  salt 
solution  to   emulsify  the  agar  cultures.     On  standardizing  such  thin 
vaccines  it  is  frequently  necessary  to  take  one  volume  of  blood  to  two, 
three,  or  even  more  volumes  of  emulsion  and  then  calculate  accordingly. 

3.  Acne  vaccine  in  20  mil.,  10  mil.  and  8  mil. 

4.  Mixed  acne  in  20  mil.  acne  and  500  mil.  staphylococcus. 

5.  Gonococcus  vaccine  in  50  mil.  and  5  mil.     Gonococcus  vaccines  are 
best  employed  as  autogenous  vaccines. 

6.  Typhoid  vaccine  in  1000  mil.  and  2000  mil.  for  prophylactic  inocu- 
lation. 

7.  Colon  vaccine  in  25  mil.,  10  mil.,  5  mil.     Vaccines  of  coliform  organ- 
isms are  very  easily  emulsified ;  as  a  rule  they  should  not  be  older  than  twelve 
hours  and  not  be  sterilized  for  more  than  three  quarters  of  an  hour. 

With  the  exception  of  the  staphylococcus  vaccines,  it  is  advisable  not  to 
use  stdck  vaccines,  but  autogenous  vaccines,  i.e.,  vaccines  made  from  the 
specific  strain  of  bacteria  causing  the  infection  to  be  treated.  It  is  very 
important  to  isolate  the  supposed  pathogenic  organism  from  the  innocuous 
or  less  pathogenic  bacteria  contaminating  or  complicating  the  infection. 

In  tuberculosis  Wright  employs  a  dilution  of  Koch  tuberculin  (T.  R.). 
Recently  he  has  prepared  a  tubercle  bacillus  vaccine  in  the  same  way  as  the 
other  bacterial  vaccines. 

The  initial  dosage  varies  with  the  different  vaccines,  but  should  in 
general  be  about  100  to  500  million  of  staphylococci;  one  may  go  as  high  as 
2,500  or  even  5,000  millions. 


212  PHAGOCYTOSIS.      OPSONINS  AND  BACTERIOTROPINS 

In  colon,  streptococcus,  gonococcus  and  acne,  doses  of  i  to  3  million 
should  be  used  at  the  beginning  and  then  gradually  increased. 

In  tuberculosis  Wright  starts  with  the  T.  R.  in  dilution  equivalent  to 
about  i/iooo  mg.  of  the  dry  tuberculin  substance  and  this  is  increased  to 
about  1/600  mg. 

Wright  cites  two  general  rules  to  be  observed  in  the  therapy  of  infectious  diseases. 

1.  In  all  cases  where  the  normal  antibacterial  power  of  the  blood  has  been  lowered, 
immunization  is  indicated. 

2.  Whenever  the  blood  possesses  strongly  active  curative  powers,  an  increase  of  the 
blood  supply  to  the  infected  part  should  be  attempted  in  order  that  the  antibacterial 
elements  of  the  blood  and  leucocytes  may  display  their   effect.     In    such   cases   the 
production  of  hyperemia  is  particularly  of  help.     Similarly,  massage  and  other  such 
therapeutic  measures  can  be  useful. 

The  therapeutic  value  of  auto-inoculation  is  very  slight  and  should  not  be  encour- 
aged, as  in  this  way  the  exact  dosage  cannot  be  followed. 

Wright  has  employed  these  vaccines  in  staphylo-,  strepto-,  and  gonococcus  infec- 
tions, as  well  as  in  coli  infections,  tuberculosis,  malta  fever  and  carcinoma  (!)  where 
injections  of  the  bacillus  neoformans  Doyen  were  given. 

From  a  critical  review  of  the  cases  published,  which  were  treated  with 
vaccines  by  Wright  and  his  fellow  workers,  one  certain  conclusion  can  be 
reached;  namely,  that  given  an  infection,  inoculations  with  small  doses  of 
the  respective  dead  or  extracted  homologous  bacteria,  will  result  in  a  thera- 
peutic immunization.  Although  Koch  had  advanced  the  same  principle 
for  the  treatment  of  tuberculosis,  it  is  Wright  who  first  recognized  the 
general  application  of  this  form  of  immunity.  Furthermore,  by  means 
of  his  opsonic  studies,  he  was  able  to  prove  that  by  the  injection  of  even 
the  minutest  doses,  for  example  1/1,000,000  c.cm.  of  tuberculin,  immune 
reactions  are  incited. 

In  spite  of  this  finding,  investigators  are  still  at  variance  over  the  ques- 
tion, and  two  camps  exist:  one  of  which  believes  that  the  ideal  treatment  of 
tuberculosis  consists  in  the  repetition  of  the  small  doses;  the  other,  that  the 
best  results  are  obtained  by  gradually  increasing  the  dose  of  tuberculin 
until  very  large  doses  are  administered.  Citron  has  found  the  latter  course 
more  satisfactory. 

Since,  as  is  known,  tuberculin  is  one  of  the  harmful  agents  in  tubercu- 
losis infections,  it  seems  more  advantageous  to  get  the  patient,  if  possible, 
into  a  condition  where  he  is  able  to  neutralize  large  doses  of  tuberculin 
rather  than  to  have  him  at  a  stage  where  even  moderate  doses  suffice  to 
give  a  reaction. 

Other  questions  of  importance  in  the  vaccine  therapy  are:  first, 
whether  any  parallelism  exists  between  the  increase  in  opsonic  index  and 
improvement  in  the  clinical  manifestations;  second,  whether  the  opsonic 
index  must  necessarily  be  used  as  a  guide  in  vaccine  treatment. 

As  to  the  first,  Wright  has  pointed  out  numerous  cases  where  exact 


VACCINE    THERAPY   AND    THE    OPSONIC    INDEX  213 

study  has  proved  that  such  parallelism  exists.  This  fact  is  probably 
correct  in  the  majority  of  instances,  but  it  cannot  be  considered  as  an  infal- 
lible rule,  inasmuch  as  the  formation  of  opsonins  is  only. one  of  a  great 
number  of  factors  in  the  complicated  process  of  healing;  consequently  one 
should  not  be  surprised  when  in  some  instances  in  spite  of  a  rising  opsonic 
index,  the  patient's  clinical  condition  becomes  worse,  and  reversely  when 
in  some  cases  improvement  occurs  although  the  opsonic  index  does  not 
change. 

Accordingly,  the  opsonic  index  during  the  course  of  treatment  becomes 
secondary  in  importance  to  the  exact  clinical  observation  of  the  case. 
Wright  and  his  school  have  shown  that  certain  bad  effects  may  follow 
from  the  injection  when  performed  during  the  negative  phase.  With 
the  use  of  small  doses  the  negative  phase  becomes  short — only  one 
day  or  even  less;  accordingly  it  is  very  probable  that  this  state  is  entirely 
passed  when  an  injection  is  repeated  on  the  fifth  to  eighth  day. 

The  tuberculin  therapy  at  the  Kraus  clinic  is  conducted  on  this  principle, 
without  estimation  of  the  opsonic  index.  And  yet,  no  harmful  effects  have 
ever  been  noted;  while  general  improvement,  as  increase  in  weight,  diminu- 
tion in  temperature  and  cessation  of  cough,  are  constantly  observed.  It 
would  be  illogical  to  neglect  these  clinical  data  and  give  preference  to  the 
hypothetical  action  of  opsonins  as  a  guide  in  treatment. 

It  seems  that  Wright  himself  does  not  insist  as  strongly  as  before  upon 
the  determination  of  the  opsonic  index.  One  of  his  assistants,  Matthews, 
has  made  the  statement  that  in  a  great  number  of  cases  the  determina- 
tion of  the  opsonic  index  is  entirely  out  of  the  question.  If  the  choice 
between  injections  without  estimation  of  the  index  and  entire  omission  of 
inoculation  should  arise,  therapeutic  inoculation  without  the  index  is  by  all 
means  indicated.  There  is  a  general  tendency  at  present  to  omit  the 
opsonic  index  in  the  treatment  of  staphylococcus  infections,  and  this  is 
at  times  also  done  in  tubercle,  gonococcus  and  streptococcus  infections  as 
well  as  in  prophylactic  typhoid  inoculations. 

Neufeld's  Method  of  Bacteriotropin  Estimation. 

Neuf eld's  technique  varies  from  that  of  Wright  in  two  points: 

(1)  He  uses  serum  free  of  complement. 

(2)  He  does  not  count  the  number  of  bacteria  within  the  leucocytes; 
but  makes  various  dilutions  of  the  serum  and  notes  in  which  dilution  the 
bacteria  are  still  ingested  in  great  numbers,  as  compared  with  a  normal 
serum  in  similar  dilution  as  control. 

Neufeld  usually  obtains  the  leucocytes  by  injecting  a  guinea-pig  intra- 
peritoneally  1 6  to  24  hours  previously  with  5  to  10  c.cm.  of  sterile  aleuronat 
solution  (i  part  aleuronat,  2  parts  bouillon).  It  is  best  to  kill  the  guinea- 
pig,  and  wash  out  the  peritoneal  exudate  which  is  full  of  leucocytes  with 


214  PHAGOCYTOSIS.      OPSONINS  AND  BACTERIOTROPINS 

40  to  60  c.cm.  of  sterile  salt  solution.  Occasionally  it  may  be  necessary  to 
use  i  1/2  per  cent,  sodium  citrate  solution  instead,  in  order  to  prevent 
coagulation.  The  leucocytes  must  be  washed  free  of  any  serum.  They 
should  not  however  be  centrifugalized  too  rapidly,  as  this  tends  to  clump 
them. 

If  rabbit's  leucocytes  are  preferred,  50  to  100  c.cm.  of  3  per  cent,  to  10 
per  cent,  peptone  bouillon  should  be  injected  intraperitoneally.  -For  mice 
i  c.cm.  aleuronat  bouillon  is  sufficient.  Human  leucocytes  are  obtained 
from  abscesses  or  from  the  blood  (Wright). 

The  serum  is  inactivated  by  heating  it  at  50°  to  60°  C.  for  one-half  hour. 
This  may  be  omitted  for  old  or  carbolized  sera  as  they  are  usually  free  of 
complement.  Also  tuberculous  sera  should  not  be  heated  as  their  bac- 
teriotropins  are  very  susceptible  to  heat. 

The  various  serum  dilutions  (i  :  10,  i  :  100,  i  :  iooo,etc.)  are  prepared 
as  usual,  but  small  quantities  suffice  since  only  0.5  to  i.o  c.cm.  of  each  dilu- 
tion is  necessary. 

The  bacteria  are  best  employed  in  the  form  of  a  16  to  24  hours  homo- 
geneous broth  culture.  If  agar  cultures  are  used,  three  loopfuls  are 
rubbed  up  in  i  c.cm.  of  salt  solution. 

For  meningococci  Neufeld  advises  agar  cultures.  Tubercle  bacilli  can  either  be 
triturated  in  an  agate  mortar  or  bought  in  the  form  of  tuberculin  residuum,  T.  R. 
(Hoechster  Farbwerke). 

Equal  parts  (0.02  to  o.i  c.cm.)  of  each  of  the  three  ingredients  (serum, 
bacteria,  leucocytes)  are  transferred  by  means  of  a  capillary  pipette,  to 
small  tubes  with  flattened  bottoms  about  4  to  5  cm.  long  and  i  cm.  wide. 
Double  the  quantity  of  leucocytes  may  be  needed  if  they  are  in  a  weak 
suspension. 

The  tubes  are  closed  by  small  corks  or  non-absorbent  cotton,  gently  but 
thoroughly  shaken  and  placed  in  the  incubator  for  definite  periods  of 
time,  depending  upon  the  micro-organism,  from  one-fourth  to  four  hours. 
The  supernatant  fluid  is  then  pipetted  off,  cover-glass  preparations  made  of 
the  sediment,  fixed  by  heat  and  stained  by  i  per  cent,  methylene-blue 
solution. 

A  great  number  of  fields  are  examined  microscopically  and  note  made  of 
the  weakest  dilution  which  still  favors  phagocytosis.  This  is  the  bacterio- 
tropic  titer  of  the  serum. 

The  necessary  controls  are:  (i)  Tube  containing  normal  serum  +  bac- 
teria +  leucocytes.  Phagocytosis  of  less  virulent  bacteria  frequently 
occurs  even  with  normal  serum.  (2)  Tube  containing  bacteria  +  leuco- 
cytes, without  any  serum;  a  virulent  bacteria  are  sometimes  taken  up  by 
leucocytes  even  without  serum.  This  is  never  the  case  with  virulent 
organisms. 


BACTERIOTROPIN   ESTIMATION    (NEUFELD)  215 

If  phagocytosis  is  entirely  absent,  one  should  not  conclude  that  bac- 
teriotropins  are  not  present.  Errors  in  technique  are  possible: 

(I)  Leucocytes  may  have  been  injured;  this  is  especially  prone  to  occur 
if  one  has  worked  with  heterologous  leucocytes;  control  examinations  with 
homologous  leucocytes  (from  same  animal  as  the  serum)  should  result  in 
phagocytosis. 

(II)  The  serum  concentration  may  be  too  high.     The  use  of  sensi- 
tized bacteria  will  obviate  this.     Bacteria  are  first  mixed  with  the  serum 
for  a  short  time,  the  mixtures  centrifugalized,  and  the  serum  pipetted  off 
leaving  a  sediment  of  sensitized  bacteria. 

(III)  The  time  during  which  the  tubes  were  in  the  incubator  may  have 
been  too  short  or  too  long.     Most  micro-organisms  require  one-half  to  two 
hours;  pneumococci  usually  need  four  hours;  cholera  vibrios  20  to  30 
minutes,  as  they  undergo  intracellular  digestion  very  readily. 

Neufeld's  technique  is  considered  by  many  simpler  than  Wright's 
method.  In  the  determination  of  normal  opsonins,  however,  concentrated 
or  only  slightly  diluted  sera  are  employed,  thus  encountering  the  difficulties 
mentioned  above  (II  and  III).  Homologous  leucocytes  and  sensitized 
bacteria  will  remove  this  interference. 

The  editor  has  found  it  puzzling  at  times  to  decide  upon  the  dilution 
injwhich  bacteriotropins  still  exist.  One  may  be  helped  in  this  decision  by 
the  presence  or  absence  of  a  great  number  of  extracellular  organisms. 


CHAPTER  XVI. 

IMMUNITY  AND  SERUM  REACTIONS  IN  REFERENCE  TO  MALIGNANT  TUMORS. 

EXPERIMENTAL    TRANSPLANTATION    OF    TUMORS.    IMMUNITY    TOWARD    TUMORS. 
SERUM  REACTIONS.     MEIOSTAGMINE  REACTION. 

The  etiology  of  malignant  tumors  is  still  unsolved.  It  has  been  defi- 
nitely proven,  however,  that  tumors  or  at  least  some  of  them  can  be  trans- 
planted. Naturally  such  experiments  with  human  cancer  are  still  limited 
and  inconclusive,  as  attempts  to  transplant  growths  from  one  person  to 
another  are  entirely  out  of  question.  On  the  other  hand,  the  inoculation  of 
human  cancer  into  lower  animals  has  been  successful  in  the  hands  of  only 
few  reliable  workers  as  Dagonet  and  C.  Lewin. 

These  failures  are  the  less  surprising  when  one  considers  that  malignant 
tumors  obtained  from  the  lower  animals,  as  rats,  and  transplanted  into 
other  animals  of  the  same  species,  not  infrequently  cease  to  grow  in  their 
new  surroundings.  Successful  implantations  of  spontaneous  animal 
rumors  vary  very  greatly,  from  4.1  per  cent.  (Bashford)  to  40  to  50  per 
cent.  (Jensen).  The  percentage  becomes  still  lower  or  even  entirely 
negative  if  the  transplantation  is  made  upon  animals  not  of  the  same 
but  of  very  closely  related  species,  for  example,  when  the  gray  house 
mouse  is  employed  instead  of  the  white  mouse. 

Once,  however,  the  tumor  continues  to  proliferate  in  its  new  host,  it 
becomes  more  easily  transplan table.  Ehrlich  has  shown  that  the  virulence 
of  a  tumor  increases  the  more  frequently  it  is  successfully  transplanted. 
Thus  a  growth  can  be  obtained  which  may  give  90  to  100  per  cent,  of  posi- 
tive grafts.  During  this  long- continued  process,  the  histological  structure 
of  the  tumor  may  change.  Not  only  may  a  carcinoma  be  transformed  into 
an  adenoma,  but  even  into  a  sarcoma,  as  was  first  observed  by  Ehrlich  and 
Apolant,  and  later  on  by  others  (Liepmann,  Bashford,  C.  Lewin). 

That  an  immunity  toward  carcinoma  may  possibly  exist  has  been 
considered. 

This  has  been  based  upon  the  facts  that  some  animals  resist  all  attempts 
at  transplantation  (" natural  immunity"),  that  in  others  the  tumor  grows 
only  to  a  limited  degree,  and  that  in  a  few,  comparatively  large  tumors  re- 
cede and  disappear  spontaneously  ("  acquired  immunity  ") .  The  last  class 
nowremains  refractory  to  all  tumors,  even  the  most  virulent,  thus  proving  a 
non-specific  immunity  ("pan  immunity").  Ehrlich  attempted  to  produce 
an  immunity  against  a  highly  virulent  mouse  carcinoma  by  using  as  vaccine 
the  hemorrhagic  mouse  tumor  which  only  rarely  allows  of  transplantation. 

216 


FREUND    KAMINER   REACTION  21 J 

Further  experiments  proved  that  a  certain  degree  of  resistance  in  an  animal 
may  be  attained  by  the  injection  of  various  cells,  be  they  embryonal  tissue 
elements  or  simply  red  blood  cells. 

Naturally  all  the  known  bacteriological  methods  for  the  destruction  of 
bacteria  have  been  applied  to  tumor  cells;  for  example  the  addition  of  dis- 
infectants, or  the  application  of  heat.  Passive  immunization  with  the 
serum  from  animals  in  whom  the  tumors  disappeared  spontaneously,  or 
from  rabbits  treated  for  a  long  time  with  increasing  quantities  of  an 
emulsion  of  carcinoma  cells,  also  gave  only  doubtful  results  even  though 
at  times  the  experiments  seemed  encouraging.  While  it  seems  probable 
that  the  growth  of  neoplasms  is  attended  by  processes  of  immunity,  one 
cannot  directly  compare  this  with  the  bacterial  or  proteid  form  of  immunity 
and  expect  the  same  antibodies  as  given  by  the  latter.  In  fact,  all  attempts 
at  a  serum  diagnosis  for  carcinoma  or  sarcoma  by  the  methods  of  precipita- 
tion or  complement  fixation  have  failed  to  withstand  careful  criticism.  The 
reactions  to  be  discussed  will,  however,  tend  to  show  that  the  sera  of  car- 
cinoma patients  possess  certain  characteristics  which  may  play  an  impor- 
tant role  in  the  future  development  of  this  problem. 

"Brieger's  cachexia  reaction"  has  been  reviewed  in  the  chapter  on  anti- 
ferments.  This  test  demonstrates  that  in  a  certain  number  of  carcinoma 
patients  the  serum  contains  greater  amounts  of  antitrypsin  than  normal 
sera.  Similarly,  many  carcinoma  sera  have  a  stronger  than  normal 
hemolysin  for  the  erythrocytes  of  the  same  or  different  animal  species  (iso- 
and  heterolysins).  Here  too  the  results  are  inconstant  and  the  sera  of 
many  noncarcinoma  patients,  especially  tuberculous,  give  similar  reac- 
tions. The  same  may  be  said  of  the  test  based  upon  the  hemolysis  of 
the  carcinoma  patients'  red  blood  cells  by  cobra  venom. 

The  Freund  Kaminer  Reaction. 

Freund  and  Kaminer  found  that  if  normal  serum  is  mixed  with  an  emul- 
sion of  carcinoma  cells  and  allowed  to  remain  at  40°  C.  for  24  hours,  the 
latter  are  broken  up  and  dissolved.  This  does  not  occur  if  the  serum 
is  derived  from  a  carcinoma  patient.  The  destruction  of  cells  is  deter- 
mined by  counting  them  in  the  Zeiss  Thoma  blood  chamber  both  before 
and  after  the  24  hours'  incubation;  or  one  may  take  the  clearing  up  of  the 
turbid  cell  emulsion  as  an  evidence  of  the  cell  destruction. 

The  technique  of  the  reaction  is  as  follows : 

Carcinoma  tissue  rich  in  cellular  elements  and  not  degenerated  is  excised  as  soon 
after  death  as  possible,  cut  up  into  small  pieces  and  placed  into  five  times  as  much  of  a 
i  per  cent,  solution  of  sodium  diphosphate.  The  whole  is  pressed  through  gauze  and 
allowed  to  stand  until  the  cells  have  sunk  to  the  bottom,  after  which  they  are  washed 
in  0.6  per  cent,  salt  solution.  They  are  again  allowed  to  settle  and  then  mixed  with 


2l8  MALIGNANT  TUMORS 

i  per  cent,  solution  of  sodium  fluoride.  The  sodium  fluoride  solution  is  first  neutralized 
(alizarin  as  indicator)  until  the  violet  color  is  reduced  to  its  minimum.  The  cell  emul- 
sion may  be  thus  preserved  for  several  weeks. 

In  the  test,  the  carcinoma  extract  is  diluted  with  0.6  per  cent,  sodium  chloride  solu- 
tion until  it  becomes  opalescent.  To  3  c.cm.  of  this  emulsion  are  added  10  drops  of 
the  patient's  serum  preferably  fresh  and  active.  They  are  allowed  to  remain  at  40°  C. 
for  24  hours  and  the  test  is  positive  if  the  turbidity  persists.  Control  tubes  must  be 
made  of  the  serum  alone,  carcinoma  extract  without  serum,  and  carcinoma  extract 
with  normal  serum.  The  last  should  become  clear. 

Recently  Freund  and  Kaminer  proposed  the  following  more  delicate  modification. 
The  supernatant  fluid  from  the  emulsion  of  carcinoma  cells  is  mixed  with  acetic  acid 
(5  c.cm.  of  acid  to  100  c.cm.  of  fluid)  heated  in  the  water  bath  for  one-quarter  of  an  hour 
at  80°  C.,  filtered,  and  after  cooling  neutralized  with  sodium  bicarbonate  against  litmus. 
Then  it  should  again  be  heated  and  filtered.  Boiling  or  heating  over  the  free  flame  is 
to  be  avoided.  An  extract  can  also  be  made  by  heating  the  tumor  itself  (preserved  in 
alcohol)  in  0.25  per  cent,  of  acetic  acid,  then  filtering  and  neutralizing.  Serum  of 
cancerous  individuals  added  to  this  extract  produces  a  cloudiness;  non-cancerous 
serum  and  extract  remains  clear.  Freund  and  Kaminer  advise  that  both  the  cell- 
counting  method  and  the  turbidity  reaction  should  be  applied  to  each  serum,  one 
acting  as  a  control  upon  the  other. 

Ranzi  and  Admiradzibi,  Kraus  and  Graff  have  corroborated  Freund 
and  Kaminer's  findings.  Rosenberg  working  under  Citron's  guidance  has 
found  that  although  in  the  main  the  reaction  is  obtained  as  above  quoted, 
some  carcinoma  sera  give  a  negative  reaction  and  some  non-carcinoma 
patients  (tuberculosis,  pregnancy)  give  a  positive  result. 


The  Meiostagmine  Reaction. 

Weichard  showed  that  by  bringing  together  antigen  and  antibodies  in 
certain  dilutions,  the  rapidity  of  diffusion  is  increased  (epiphanin  reaction). 
M.  Ascoli  further  demonstrated  that  the  union  between  a  specific  antigen 
and  its  specific  serum  is  associated  with  appreciable  lowering  in  the  surface 
tension,  so  that  the  number  of  drops  to  a  definite  quantity  of  fluid  is  dis- 
tinctly increased  (Meiostagmine  reaction).  This  term  is  of  Greek  deriva- 
tion, "paw,"  smaller,  "o-ray/wx,"  drops.  Traube's  "Stalagmometer"1 
measures  the  number  of  drops. 

The  meiostagmine  reaction  has  been  tested  by  Izar  and  Vigano  in 
typhoid,  paratyphoid  and  lues  and  found  to  possess  a  certain  degree  of 
specificity.  In  tuberculosis,  the  test  is  positive  only  in  active  cases. 
Ascoli  and  Izar  claim  to  get  reliable  results  with  their  method  also  in  car- 
cinoma. Their  technique  is  as  follows: 

1  The  Stalagmometer  of  Traube  is  merely  a  very  finely  and  elaborately  graduated 
pipette  with  a  central  bulbous  reservoir.  The  dropping  end  of  the  instrument  ends 
in  a  flattened  ground  base  thus  insuring  uniformity  in  the  size  of  the  drops.  The 
instrument  is  so  graduated  that  a  fraction  of  a  drop  can  be  estimated. 


THE   MEIOSTAGMINE   REACTION 


219 


i.  Extract:  a  malignant  tumor  which  has  not  undergone  degeneration 
(or  a  sheep's  pancreas)  is  cut  into  fine  slices  or  small  pieces.  It  is  spread 
out  on  glass  plates  and  dried  in  a  temperature  of  37°  C.  The  dried  residue 
is  powdered  and  extracted  with  methyl  alcohol  in  the  proportion  i  :  4  for 
24  hours  at  50°  C.  The  extract  is  filtered  through  a  hard  filter,  at  first 
hot  and  then  cold.  It  should  be  protected  from  the  light,  but  should  not 
be  placed  in  the  ice-box.  The  dilutions  cannot  be  preserved,  but  must  be 
made  fresh  every  time,  by  measuring  out  the  requisite  amount  of  extract 
with  a  dry  pipette,  placing  it  in  a  dry  test-tube  or  flask,  and  adding  the 
required  quantity  of  distilled  water  and  shaking  thoroughly. 

The  extract  is  first  titrated:  dilutions  1 150,  i  175,  i  :ioo,  i  1125,  etc., 
are  made,  and  to  i  c.cm.  of  each  of  these,  9  c.cm.  of  a  i  :  20  normal  serum 
diluted  with  saline  are  added.  The  mixtures  are  placed  for  two  hours  at 
37°  C.  The  number  of  drops  to  each  quantity  is  estimated.  A  control 
tube  containing  the  same  quantity  of  normal  serum  plus  i  c.cm.  of  distilled 
water  is  also  counted.  That  dilution  is  chosen  as  the  working  dose  which 
differs  from  the  control  tube  by  3  to  5  fractions  of  a  drop. 

The  test  is  performed  as  follows:  To  9  c.cm.  of  each  serum  diluted  with 
saline  i  :  20  is  added  i  c.cm.  of  distilled  water  and  the  number  of 
drops  falling  from  the  stalagmometer  is  counted  before  and  after  incu- 
bating for  two  hours  at  37°  C.  or  one  hour  at  50°  C.  This  is  used  as  a 
control.  To  9  c.cm.  of  each  serum  diluted  i  :  20  with  normal  saline  is 
added  i  c.cm.  of  antigen  emulsion  of  the  desired  strength,  e.g.,  dilution 
i  :i25.  The  number  of  drops  in  this  mixture  is  counted  only  after 
incubation.  As  further  controls  it  is  advisable  to  have  several  known 
normal  and  tumor  sera.  The  reaction  is  positive  if  the  number  of  drops 
contained  in  the  mixture  of  tumor  extract  and  tumor  serum  is  greater  by 
at  least  11/2  drops  than  in  the  controls  after  the  incubation. 


9  c.cm.  serum  i  /2O 


Number  of  drops  per  c.cm. 
with  i  c.cm.  tumor  extract 

1:125 
(after  incubation) 


Number  of  drops  per  c.cm. 

with  i  c.cm.  of  distilled  water 

(after  incubation) 


Normal  serum  

58+41 

58+2 

Normal  serum  .  .  . 

59 

58+7 

Carcinoma  serum 

61+6 

58+8 

Carcinoma  serum  

60+4 

58+5 

The  antigens  spoil  very  easily.  Recently,  chemicals  in  the  form  of  lip- 
oids  are  being  substituted  for  the  specific  antigen.  This  simplifies  matters 
greatly.  In  looking  over  the  numerous  publications  by  Ascoli  and  his 

1S8+4  means  58  drops  +  4  fractions  of  a  drop. 


220  MALIGNANT  TUMORS 

followers,  one  meets  with  constant  variations  in  the  technique  of  the 
reaction.  Thus  it  is  very  difficult  to  arrive  at  an  exact  description  or^a 
conclusive  opinion.  Many  non-cancerous  sera  give  a  positive  meiostag- 
mine  reaction.  The  test  has  not  yet  been  adopted  as  a  clinical  aid. 
A  review  of  the  early  literature  of  this  subject  is  given  by  Bernstein 
and  Simons,  Amer.  Jour.  Med.  Sciences,  Dec.,  1911. 


CHAPTER  XVII. 
ANAPHYLAXIS. 

Introduction.   Proteid  Hy  per  susceptibility .  Anaphylaxis.  Passive  Anaphy- 
laxis.  Anaphylatoxin.  Serum  Disease.  Hay  Fever. 

The  explanation  for  many  phenomena  discussed  in  the  former  chapters 
was  based  upon  the  observation  that  by  overcoming  an  infectious  disease 
the  organism  undergoes  some  transformation  whereby  it  becomes  refrac- 
tory or  less  susceptible  to  the  same  infection.  This  changed  state,  demon- 
strated neither  by  chemical  nor  microscopical  methods,  was  termed 
"Immunity."  The  most  constant  evidence  of  immunity  is  the  presence 
of  antibodies.  This,  however,  should  by  no  means  imply,  as  is  so  often 
done  in  literature,  that  the  demonstration  of  antibodies  and  the  exist- 
ence of  immunity  are  identical. 

On  the  contrary,  attention  has  already  been  called  to  the  possibility  of 
paradoxical  reactions.  Animals  or  individuals  in  whom  a  grade  of  immu- 
nity is  expected,  show  instead  a  certain  susceptibility  toward  the  particu- 
lar antigen.  This  altered  reaction  of  the  organism  was  termed  "Hyper- 
susceptibility"  and  "Anaphylaxis"  (to  distinguish  it  from  prophylaxis- 
immunity).  The  "paradoxical  reaction"  was  discussed  under  diphtheria 
where  it  was  mentioned  that  horses  immunized  for  a  long  period  of  time 
would  suddenly  become  severely  ill  after  the  injection  of  small  doses  of 
toxin.  The  tuberculin  reaction  was  another  instance  of  such  hypersensi- 
tiveness.  Infection  by  the  tubercle  bacillus  not  only  made  the  body  highly 
sensitive  toward  the  bacteria  and  their  derivatives  (tuberculin),  but  a 
severe  and  characteristic  reaction  always  took  place  as  the  expression  of 
this  increased  sensitiveness. 

In  recent  years,  experimental  studies  have  proven  that  this  peculiar 
phenomenon  is  not  based  upon  entirely  new  laws,  but  that  anaphylaxis 
has  as  its  governing  influences  principles  that  are  closely  related  to  those 
influencing  the  process  of  immunity.  It  is  still  to  be  determined  whether 
the  hypers usceptibility  observed  with  various  antigens  like  the  pure  toxins, 
the  tuberculins,  and  the  proteids  all  follow  the  same  biological  mechanism. 
The  author  is  of  the  opinion  that  just  as  there  are  various  forms  of  immu- 
nity so  also  must  the  existence  of  various  forms  of  anaphylaxis  be  presumed. 
At  least  a  cellular  and  humoral  variety  seem  distinctly  plausible;  the 
tuberculin  reaction  is  an  example  of  the  former,  the  proteid  anaphylaxis 
of  the  latter. 

221 


222  ANAPHYLAXIS 

The  parenteral  introduction  of  a  foreign  proteid  into  an  or- 

Proteid      ganism  results  in  the  formation  of   antibodies   against  this 

Anaphylaxis.  proteid:  precipitins,  cytolysins,  complement  fixation  bodies, 

etc.  At  first  it  was  assumed  that  a  "proteid  immunity"  had 
taken  place.  Such  conclusion  proved  erroneous. 

Richet  and  Portiers  (1902)  in  working  with  actino-congestin  (a  substance  extracted 
from  the  tentacles  of  Actinia)  demonstrated  that  dogs  injected  with  sublethal  doses  of 
this  toxin  would  die  acutely  if  the  injection  were  repeated  after  an  interval  of  three 
weeks.  In  the  light  of  more  recently  established  facts,  the  explanation  of  Richet's 
work  was  complicated  because  actino-congestin  consists  of  two  components,  a  true 
toxin  against  which  an  immunity  can  be  stimulated,  united  with  a  proteid  element 
which  like  all  proteids  produces  anaphylaxis. 

It  was  therefore  of  extreme  fundamental  importance  when  Arthus  and 
Theobald  Smith  showed  that  proteid  substances,  of  themselves  apparently 
non-toxic,  can  constantly  produce  the  phenomena  associated  with  hyper- 
sensitiveness. 


The  Arthus  Phenomenon. 

If  a  rabbit  is  injected  subcutaneously  with  horse's  serum  at  intervals  of 
six  days,  a  soft  infiltrate  which  remains  for  two  to  three  days  appears  at 
the  site  of  injection  after  the  fourth  inoculation,  a  harder  infiltration  which 
continues  for  a  longer  period  of  time  after  the  fifth  inoculation,  and  gangrene 
after  the  sixth  or  seventh.  A  rabbit  repeatedly  treated  intravenously 
with  horse's  serum  may  die  with  severe  general  symptoms  several  minutes 
after  one  of  the  later  inoculations. 

The  Theobald  Smith  Phenomenon. 

Theobald  Smith  observed  that  guinea-pigs  injected  with  neutral  mix- 
tures of  diphtheria  toxin  and  horse's  antitoxic  serum  would  be  killed  if 
after  an  interval  of  several  weeks  they  were  given  a  subcutaneous  injection 
of  normal  horse's  serum. 

Otto  and  others  showed  that  both  of  these  phenomena,  above  described, 
are  identical  in  their  principle;  thus,  that  of  Arthus  can  be  likewise  in- 
duced after  a  single  injection  of  horse's  serum  if  the  first  dose  is  small,  and 
if  the  interval  between  the  first  and  second  inoculation  is  sufficiently  long 
(about  three  weeks  or  more). 

Further  study  proved  that  anaphylaxis  may  be  incited  by  repeated  paren- 
teral introduction  of  practically  any  foreign  proteid. 

The  first  inoculation  prepares  the  animal  in  such  a  manner  that  after  a 
definite  incubation  period  the  second  injection  of  the  same  serum  will 


PRINCIPLES   OF   ANAPHYLAXIS  223 

bring  on  characteristic  acute  symptoms  which  may  terminate  fatally. 
The  picture  of  hypersensitiveness  or  " serum  sickness"  in  man,  as  described 
by  v.  Pirquet  and  Schick,  is  classical.  Other  investigators  who  deserve 
merit  for  work  in  this  field  are  Arthus,  Otto,  Rosenau,  Anderson,  Kraus, 
Doerr,  Besredka,  Weichardt,  Wolff-Eisner,  Friedemann,  Friedberger,  H. 
Pfeiffer,  Schittenhelm. 

It  is  through  their  efforts  that  the  close  relationship  between  hyper- 
sensitiveness  and  immunity  is  more  clearly  understood.  Like  the  state  of 
immunity,  anaphylaxis  is  either  spontaneous  or  acquired.  It  is  also  specific, 
that  is  a  guinea-pig  made  sensitive  toward  horse's  serum  will  react  only 
when  again  treated  with  horse's  serum  but  not  when  receiving  rabbit's  or 
human  serum. 

A  classical  anaphylaxis  experiment  may  be  carried  out  as  follows.  A 
guinea-pig  receives  a  subcutaneous  injection  of  o.ooi  to  o.oi  c.cm.  of  horse 
serum  and  after  three  weeks  an  intravenous  injection  of  3  to  5  c.cm.  is 
repeated.  This  quantity,  which  under  normal  conditions  has  no  influence 
upon  the  animal,  will  now  produce  very  alarming  symptoms  or  even  death 
in  a  couple  of  minutes. 

There  are  several  factors  upon  which  the  occurrence  of  the  anaphylactic 
phenomena  strictly  depends. 

(a)  The  first,  preparatory,  or  sensitizing  dose  must  enter  the  system 
in  some  way  other  than  through  the  gastro-intestinal  tract.     Only  ex- 
ceptionally does  hypersensitiveness  arise  if  the  antigens  are  given  per  os. 

(b)  The  quantity  of  antigen  is  of  the  utmost  importance.     The  smallest 
amounts  of  proteid  suffice,  e.g.,  o.oooooi  c.cm.  serum.     As  aruleo.ooi  to 
o.oi  c.cm.  are  employed. 

(c)  An  incubation  period  (the  preanaphylactic  state)  is  always  neces- 
sary.    This  period  varies  in  the  widest  degree,  but  depends  primarily 
upon  the  amount  of  antigen  first  injected. 

With  guinea-pigs,  it  is  never  less  than  seven  days.  If  o.oi  to  o.i  c.cm.  serum  is 
injected,  symptoms  may  usually  be  stimulated  after  ten  days.  Very  large  doses  as 
well  as  very  minute  ones  increase  the  length  of  the  preanaphylactic  state  very  mark- 
edly (as  long  as  three  months). 

(d)  The  susceptibility  of  the  various  animal  species  differs  greatly. 
The  most  suitable  is  the  guinea-pig,  the  rabbit  far  less  so. 

(e)  The  actual  anaphylactic  shock  is  dependent  upon  the  quantity  of 
the  second  dose  injected  and  the  mode  of  injection.     The  intravenous  path 
of  a  massive  dose  is  the  most  reliable;  then  comes  the  intraperitoneal  and 
then  the  subcutaneous  method.     While  with  the  intravenous  procedure 
the  final  result  is  usually  death,  this  is  almost  never  so  with  the  subcuta- 
neous injection.     Here  the  anaphylaxis  expresses  itself  in  a  more  marked 
local  inflammation,  edema  and  eventually  necrosis. 


224  ANAPHYLAXIS 

An  animal  that  recovers  from  the  second  injection  becomes 

Anana-      immune  to  further  administration  of  the  same  proteid.     This 

phylaxis.     non-susceptible  condition  has  been  wrongly  termed  antiana- 

phylaxis.  It  is  much  more  reasonable  to  speak  of  "anana- 
phylaxis,"  since  the  absence  of  hypersensitiveness  is  not  due  to  the  neu- 
tralization of  the  bodies  necessary  for  bringing  about  the  hypersuscepti- 
bility.  The  ananaphylactic  state  sets  in  as  early  as  two  hours  after 
the  anaphylactic  outburst. 

In  order  to  prevent  the  shock  of  anaphylaxis,  it  has  been  suggested  by 
Besredka  and  Steinhardt  to  give  the  second  injection  during  the  period  of 
incubation,  i.e.)  about  the  eighth  day,  or  give  a  very  minute  dose  of  serum 
at  the  regular  time  of  the  second  inoculation  and  give  the  larger  dose  in 
24  hours. 

Just  as  an  immunity  may  be  transmitted  by  injecting  the  serum 

Passive     obtained  from  an  immune  animal,  so  also  can  the  tendency 

Anaphylaxis.  to  hypersusceptibility  be  transmitted  by  introducing  into  a 

normal  animal  the  serum  from  a  sensitized  one,  i.e.,  one  that 
has  been  injected  with  a  foreign  proteid.  This  is  best  demonstrated  by 
injecting  the  anaphylactic  serum  subcutaneously,  followed  in  24  hours 
by  the  intravenous  inoculation  of  the  respective  antigen. 

A  fully  satisfactory  explanation  for  all  the  phenomena  of 
Theories     anaphylaxis  has  not  as  yet  been  advanced.     Certain  it  is 
of  Anaphy-    that  a  number  of  underlying  factors  exist  which  bring  ana- 
laxis.        phylaxis  and  immunity  into  close  relationship. 

Since  the  term  immunization  usually  implies  a  beneficial 
process,  while  anaphylaxis  in  most  instances  represents  a  situation  of  an 
injurious  nature,  v.  Pirquet  recommended  the  term  " allergic"  to  designate 
the  reactive  changes  which  an  organism  generally  exhibits  after  infection 
or  injection  of  an  antigen.  The  allergic  phenomena  are  divided  into 
those  associated  with  diminished  susceptibility,  i.e.,  prophylaxis,  and  those 
with  increased  sensitiveness,  i.e.,  anaphylaxis. 

Besredka  adheres  to  the  view  that  the  anaphylactic  syndrome  expresses  an  insult  to 
the  central  nervous  system.  He  was  able  to  show  that  susceptible  guinea-pigs,  when 
etherized,  will  bear  the  second  inoculation  of  the  serum  perfectly  well.  V.  Pirquet  and 
Schick  consider  the  precipitin  action  as  the  basis  for  the  anaphylactic  phenomena.  In 
the  main,  however,  there  are  two  theories,  a  cellular  and  a  humoral  one.  The  former 
suggests  that  the  hypersusceptibility  is  due  to  the  stimulation  of  new  specific  receptors 
which  remain  sessile,  i.e.,  attached  to  the  body  cells  instead  of  being  thrown  off  into 
the  blood  stream.  When  more  antigen  is  injected,  these  cells,  due  to  their  greater 
affinity,  absorb  more  of  the  toxic  substance  of  the  antigen  than  they  do  normally,  and 
thus  the  anaphylactic  shock  is  incited. 

The  humoral  theory  represents  the  main  activity  within  the  serum.  This  hy- 
pothesis was  adopted  by  Wolff-Eisner,  a  pupil  of  Pf eiffer,  and  was  based  upon  Pfeiffer's 
endotoxin  principle.  It  takes  for  granted  that  all  antigens,  cells  and  proteids,  contain 


ANAPHYLATOXIN  225 

within  their  bodies  a  toxic  substance  that  does  not  form  antibodies  when  liberated  in 
animals.  The  first  injection  of  antigen  produces  bacteriolytic  or  cytolytic  antibodies 
possessing  the  power  of  liberating  the  endotoxic  poisons  from  the  proteid  molecule. 
When  the  second  injection  is  given,  these  bacteriolytic  antibodies  at  once  cause  a  rapid 
liberation  of  the  intracellular  toxic  fraction,  and  injury  to  the  animal  results.  Wolff- 
Eisner's  theory  can  apply  only  in  a  certain  number  of  instances,  because  the  essential 
factor  of  cytolysis  is  not  always  present;  very  many  bacteria,  especially  tubercle  bacilli, 
are  not  thus  broken  up. 

This  view  has,  however,  been  the  fundamental  thought  for  the  later  very  important 
work  of  Friedemann  and  Friedberger.  By  these  supporters  of  the  humoral  theory 
the  main  influence  is  placed  upon  the  union  which  takes  place  in  vivo  between  the  anti- 
gen -f-  amboceptor  +  complement.  As  a  result,  anaphylaxis  may  be  incited  in  one  of 
two  ways.  The  mere  absorption  of  the  complement  may  bring  about  the  anaphylactic 
shock.  This  diminution  in  the  complement  content  of  the  serum  is  always  demon- 
strable. Or,  as  is  more  probable,  the  union  of  the  above  three  elements  causes  a  destruc- 
tion of  the  antigen  and  a  liberation  of  a  toxic  agent.  This  theory  is  strengthened  by  the 
demonstration  that  not  only  in  vivo  but  also  in  vitro  can  such  a  toxic  substance  be 
obtained  by  mixing  antigen  -f-  amboceptor  +  complement. 

In  1902  Weichardt  immunized  rabbits  with  a  proteid  derived 
from  syncytial  cells.     By  mixing  the  fresh  immune  serum 
with  an  emulsion  of  the  syncytial  cells  and  filtering,  he  derived 
a  very  toxic  fluid  which  was  named  syncytio toxin.     In  1909 
Friedemann  and  Citron  simultaneously  isolated  in  vitro  toxic  elements 
which  could  bring  about  anaphylaxis.     This  was  confirmed  by  Fried- 
berger several  months  later.     Friedemann  got  his  poisonous  agent  by 
mixing  erythrocytes,  inactive  hemolytic  serum  and  complement. 

Three  c.cm.  sheep's  blood  +  i  c.cm.  inactive  hemolytic  serum  are  allowed  to 
remain  at  room  temperature  for  one-half  hour,  then  centrifugalized.  The  sensitized 
erythrocytes  are  mixed  with  4  c.cm.  fresh  rabbit's  serum  (complement)  and  placed 
into  the  water  bath  at  37.5°  C.  for  exactly  five  minutes;  then  cooled  down  by  ice  and 
centrifugalized.  The  red  blood  cells  remain  almost  all  unhemolyzed.  The  superna- 
tant fluid  is  only  slightly  reddened  and  when  injected  intravenously  into  a  rabbit 
(1310  gms.)  causes  general  weakness,  diarrhea  and  death  on  the  following  day. 

It  is  to  be  observed  that  the  anaphylatoxic  substance  was  formed  even 
though'  no  hemolysis  took  place. 

Citron  accomplished  the  same  end  by  a  mixture  of  tuberculin,  serum 
from  a  tuberculous  individual  who  spontaneously  produced  antituber- 
culin  amboceptors,  and  normal  guinea-pig's  serum  (complement). 

o.i  to  0.2  c.cm.  T.+  0.2  to  0.4  c.cm.  antituberculin  +  0.3  to  0.5  c.cm.  comple- 
ment are  mixed  and  incubated  for  one  hour.  The  entire  quantity  is  injected  intra- 
peritoneally  into  tuberculous  guinea-pigs;  the  animals  die  in  four  to  five  hours. 
Tuberculous  animals  receiving  o.i  to  0.2  c.cm.  T.  alone  or  o.i  to  0.2  c.cm.  T.  -f  0.2  to 
0.4  c.cm.  antituberculin,  remain  alive. 


226  ANAPHYLAXIS 

Friedberger  demonstrated  that  a  toxic  product  which  he  named  "Ana- 
phylatoxin"  can  be  obtained  in  vitro  from  every  proteid,  be  it  animal  or 
bacterial  in  nature.  His  technique  is  almost  identical  with  that  of  Friede- 
mann  described  above. 

Accordingly,  Friedberger  proposed  a  theory  explaining  the  course  and 
nature  of  all  infectious  diseases  upon  the  basis  of  such  a  common  anaphy- 
latoxin, independent  of  the  character  and  virulence  of  the  bacteria. 
While  this  hypothesis  takes  into  consideration  the  older  belief  that  disease 
is  due  to  a  struggle  between  the  infectious  agent  and  the  invaded  organism, 
it  does  so  in  a  somewhat  broader  sense.  From  Friedberger's  standpoint 
it  is  the  unspecific  anaphylatoxin  which  is  the  important  factor;  its  vari- 
able quantity,  which  stands  in  direct  association  with  the  variable  pro- 
portions of  antigen,  amboceptor  and  complement,  accounts  for  the 
various  pictures  of  the  different  bacterial  infections. 

This  explanation  hardly  suffices  for  all  the  characteristic  symptoms  of 
the  infectious  diseases.  It  does,  however,  account  for  one,  the  fever. 

That  some  relationship  exists  between  hypersusceptibility  and  the 
thermal  center  was  first  observed  by  H.  Pfeiffer  and  Mita.  The  ana- 
phylactic  shock  is  always  associated  with  a  fall  in  temperature,  and  at 
times  this  may  be  its  only  manifestation. 

A  different  effect  upon  the  temperature  results  according  to  the  dif- 
ferent quantity  of  anaphylatoxin  injected  (Schittenhelm  and  Weichardt, 
Friedberger  and  Mita).  Well-defined  limits  have  been  established. 

Aside  from  the  effect  upon  the  temperature,  other  characteristic 
phenomena  of  the  anaphylactic  intoxication  are  the  incoagulability 
of  the  blood  and  the  marked  distention  of  the  lungs  as  found  by  post 
mortem  examination. 

The  symptoms  of  active  anaphylaxis  differ  in  the  various  ani- 

Serum      mals.     In  man  very  exact  studies  of  the  results  of  the  repeated 

Disease,     injections  of  foreign  serum  have  been  made  by  V.  Pirquet  and 

Schick. 

The  evidences  of  serum  sickness  are  numerous.  Those  which  are  pres- 
ent most  frequently  are  fever,  skin  eruptions,  swelling  of  the  joints,  glan- 
dular enlargement  and  edema. 

These  symptoms  may  follow  even  the  very  first  injection  of  serum.  As 
a  rule  they  develop  after  an  incubation  period  of  eight  to  ten  days.  Slight 
reddening  at  the  point  of  injection  accompanied  by  moderate  swelling  of 
the  regional  lymph  glands  appear  as  prodromal  manifestations. 

The  general  condition  of  the  patient  is  generally  only  very  little  dis- 
turbed in  spite  of  the  frequently  associated  high  fever.  Still  there  are 
instances,  especially  after  the  introduction  of  large  amounts  of  serum, 
where  the  symptoms  continue  for  about  four  to  five  weeks  and  then  lead  to 
severe  disturbances. 


SYMPTOMS   OF   SERUM   SICKNESS  227 

The  associated  skin  eruptions  are  usually  of  the  type  of  an  urticaria; 
although  Hartung  describes  rashes  simulating  scarlet  fever  and  measles. 

V.  Pirquet  and  Schick  consider  the  following  as  the  most  positive 
symptoms  of  serum  sickness: 

1.  The   occurrence   of  the  exanthema  seven   to  fourteen  days  after 
injection. 

2.  First  appearance  of  the  rash  around  the  point  of  injection. 

3.  Regional  enlargement  of  the  lymph  glands. 

4.  Complete  absence  of  any  changes  in  the  mucous  membrane. 
Measles  is  excluded  by  the  absence  of  Koplik  spots,  coryza,  and  con- 
junctivitis. 

In  scarlet  fever  the  following  symptoms  help  to  exclude  serum  sickness: 

1.  Initial  vomiting. 

2.  Occurrence  of  angina  before  or  at  the  same  time  as  the  exanthema. 

3.  High  fever. 

4.  The  simultaneous  existence  of  the  infection  among  others  in  the 
hospital  or  neighborhood. 

If  the  serum  disease  does  not  arise  after  the  first,  but  after  a  later  in- 
jection, it  is  characterized  by  the  absence  of,  or  very  marked  diminution  in, 
the  length  of  the  period  of  incubation,  and  in  addition  by  increased  severity 
of  the  symptoms. 

In  the  dog  these  phenomena  have  been  carefully  observed  by 

Anaphylaxis  Biedl  and  Kraus  and  Arthus:  3  to  5  c.cm.  of  horse  serum  is 

in  the  Dog.  administered  for  the  first  injection  and  after  three  weeks  10 

c.cm.  are  given  intravenously.     In  about  one-half  a  minute  the 

dog  becomes  restless,  begins  to  vomit,  and  has  involuntary  evacuation  of 

urine  and  feces.    This  is  followed  by  a  period  of  excessive  prostration  during 

which  the  dog  lies  with  his  limbs  outstretched  and  almost  motionless  as  if 

paralyzed.     After  several  hours  the  animal  either  begins  to  recover  or  dies. 

Biedle  and  Kraus  noticed  that  about  15  to  30  seconds  after  the  intra- 
venous reinjection,  the  arterial  blood  pressure  begins  to  sink  rapidly.  This 
has  been  ascribed  to  the  wide  dilatation  of  the  peripheral  blood  vessels  due 
to  a  paralysis  of  the  peripheral  vasomotor  system.  In  addition  there  is  a. 
leucopenia  and  a  diminution  in  the  coagulation  power  of  the  bloodr 
symptoms  which  have  been  observed  also  in  man.  The  animal  par  ex- 
cellence for  anaphylaxis  experiments  is  the  guinea-pig.  The  rabbit  is 
the  next  choice. 

If  guinea-pigs  receive  as  their  second  inoculation  a  large  dose  of  serum  in- 
Anaphylaxis  travenously,  they  die  acutely  (Th.  Smith) ;  (animals  that  have  not  been 
in  the       sensitized  bear  this  same  quantity  of  serum  without  any  disturbance- 
Guinea-pig    whatsoever).     The  blood  pressure  first  rises,  then  sinks  rapidly.     At 
and  Rabbit,    postmortem   the  lungs    are    firmly    inflated    (Gay    and    Southardt)> 
Death  is  probably  caused  by  respiratory  paralysis,  Auer  and  Lewis 
having  described  a  tetanic  spasm  of  the  bronchial  muscles. 


228  ANAPHYLAXIS 

If  for  the  second  injection  only  a  small  dose  of  serum  is  administered,  or  it  is  intro- 
duced intraperitoneally  instead  of  intravenously,  acute  death  does  not  result  and  the 
picture  is  similar  to  that  given  by  the  anaphylactic  dog  (Pfeiffer  and  Mita).  The 
guinea-pigs  become  very  restless,  move  about  continuously  and  are  very  easily  fright- 
ened. Their  hair  becomes  raised,  and  isolated  clonic  muscular  contractions  may  be 
observed.  Continuous  hiccough  sets  in  and  evacuation  of  urine  and  f eces,  first  formed 
and  later  fluid  occurs.  The  abdominal  muscles  become  spastically  rigid  and  at  times  a 
severe  pruritus  of  the  skin  probably  exists.  Following  this  transient  period  of  excita- 
tion the  animals  enter  into  a  stage  of  depression.  They  stagger  about  or  fall  to  one  side 
and  remain  for  hours  as  if  paralyzed;  breathing  is  slow  and  superficial.  In  the  midst  of 
these  symptoms  the  temperature  falls  abruptly,  often  as  many  as  7°  to  13°  C.  Death 
is  due  to  a  paralysis  of  the  peripheral  blood-vessels  and  takes  place  usually  in  one  to 
two  hours,  sometimes  four  to  eight  hours,  being  always  preceded  by  Cheyne-Stokes 
respiration. 

The  symptoms  of  the  anaphylactic  shock  simulate  very  closely 
Relation  to  those  occasioned  by  peptone  poisoning,  as  seen  after  intra- 
Peptone     venous  injection  of  Witte's  peptone,  for  example.     Biedl  and 
img'   Kraus  thus  considered  that  anaphylaxis  was  a  manifestation 
of  peptone  poisoning.     They  believed  that  through  immunization  with 
proteids,  antibodies  of  a  ferment  nature  were  stimulated,  and  that  these 
split  up  the  proteids  into  peptones.     According  to  these  authors,  animals 
exposed  to  peptone  poisoning  become  hypersensitive,  and  vice  versa, 
animals  that  recover  from  an  anaphylactic  attack  withstand  poisoning  by 
peptones.     The  production  of  anaphylatoxin  in  vitro,  and  the  demonstra- 
tion by  Pfeiffer  that  peptone  is  formed  in  the  test-tube  during  this  process, 
further  added  to  the  support  of  their  theory. 

This  hypothesis  cannot  as  yet  be  definitely  accepted.     In 
Toxopeptid.  the  first  place  Manwaring  was  unable  to  confirm  the  experi- 
ment that  recovery  of  an  animal  from  an  anaphylactic  shock 
renders  it  refractory  toward  future  peptone  poisoning.       Furthermore, 
M.  Wassermann  and  Keysser  mixed  kaolin  +  inactive  immune  serum  + 
complement  and  obtained  a  poison  which  caused  the  same  disturbances  as 
anaphylatoxin.     Since  kaolin  is  no  proteid  and  cannot  be  split  up,  the 
anaphylactic  symptoms  in  this  instance  cannot  be  due  to  a  peptone  as  a 
split  product  of  the  antigen. 

M.  Wassermann  and  Keysser  have  a  different  conception  of  the  nature  of  anaphy- 
laxis. The  antigen  serves  in  a  physical  chemical  capacity  to  fix  the  amboceptor.  By 
the  addition  of  complement  the  amboceptor  is  broken  up  with  the  formation  of  "Toxo- 
peptids,"  and  it  is  these  which  stimulate  the  anaphylactic  phenomena.  Passive 
anaphylaxis  is  explained  by  the  passive  transmission  of  the  specific  amboceptors;  the 
antigen  itself  plays  no  chemical  role;  it  is  not  split  up.  The  difference  between  ana- 
phylaxis and  immunity  lies  in  that  in  the  former  the  complement  under  cover  of 
the  antigen  digests  the  amboceptor  alone,  while  in  the  state  of  immunity  the  strength 
and  number  of  amboceptors  are  very  much  greater  and  the  activity  of  the  complement 
extends  not  only  to  the  amboceptor  but  also  to  the  secondarily  attached  antigen. 


HAY-FEVER  229 

It  would  be  impractical  to  discuss  all  the  theories  enlisted  for  anaphy- 
laxis.  Exact  facts  are  stil]  insufficient.  Experimental  work  is  constantly 
disclosing  new  ideas. 

More  detailed  reviews  may  be  found  in  the  following  articles: 

H.  Pfeiffer:  Problem  of  Pro teid  Anaphyl axis.     G.  Fischer,  Jena,  1910. 

A.  Schittenhelm:  Anaphylaxis  from  the  standpoint  of  pathological 
physiology  and  the  clinic.  Jahresbericht  iiber  die  Ergebnisse  der  Immun- 
itats  forschung.  Enke,  Stuttgart,  1910. 

J.  Citron:  Critical  review  of  the  problem  of  anaphylaxis.  Fol.  sero- 
logica,  1911,  Bd.  vii,  H.  3. 

Friedberger:  Anaphylaxis.  Fortschritte  der  deutschen  Klinik.  Bd.  ii. 
Urban  u.  Schwarzenberg,  Berlin- Wien,  1911. 

H.  Bold:  Bacterial  Anaphylatoxin  and  its  Importance  in  Infections. 
G.  Fischer,  Jena,  1912. 

Besides  bacterial  infections,  recent  teaching  places  urticaria, 
Hay-fever,    eclampsia,  bronchial  asthma  and  hay-fever  into  the    class 
of  diseases  with  an  anaphylactic  basis.     This  is  especially 
applicable  to  hay  fever,  formerly  considered  a  pure  intoxication,   the 
pollen  toxin  having  been  described  by  Dunbar  as  the  etiological  factor. 
In  Germany  the  disease  seems  to  come  chiefly  from  pollen  of  the  grasses 
and  grains   (rye  pollen  being  most   active);   whereas   in  America,    ap- 
parently,   the   most   important  pollen   springs  from  the  ambrosia  (rag 
weed),   solidago  (golden  rod),  and  other  members  of  the  family  of  the 
compositae. 

The  toxin  is  isolated  by  mixing  for  ten  hours  the  ground  pollen  with 
5  per  cent.  NaCl  solution  ard  0.5  per  cent,  phenol  at  37°  C.  Then, 
in  the  form  of  a  proteid  it  is  precipitated  by  the  addition  of  eight  to  ten 
volumes  of  96  per  cent,  alcohol  and  the  resultant  white  precipitate  dis- 
solved in  physiological  salt  solution. 

Susceptibility  to  the  pollen  toxin  is  limited  only  to  certain  individuals. 
Some  are  influenced  by  the  rye  pollen  only,  others  by  the  golden  rod  alone, 
while  a  third  class  is  affected  by  all.  The  cause  for  this  peculiar  idiosyn- 
crasy is  unknown.  The  majority  of  observers  are,  however,  now  agreed 
that  one  is  dealing  here  with  a  reaction  of  hypersusceptibility,  as  was  first 
pointed  out  by  Weichardt  and  Wolff- Eisner.  Only  by  means  of  antibodies 
does  this  non-toxic  pollen  proteid  become  a  poison. 

All  those  who  suffer  from  hay-fever  develop  a  marked  conjunctivitis 
whenever  even  the  slightest  amount  of  pollen  proteid  (i/iooo  mg.)  is 
dropped  into  the  conjunctival  sac.  In  addition,  all  the  symptoms  of  hay- 
fever  or  asthma  may  be  incited.  Sim^ar  effects  are  in  evidence  when  sub- 
cutaneous injections  are  resorted  to. 

For  purposes  of  immunization  horses  are  most  suitable,  but  only  those 
which  after  an  injection  of  pollen  extracts  manifest  a  local  and  general  re- 


230  ANAPHYLAXIS 

action.  This  is  found  in  one-third  of  the  animals.  Their  serum  thus 
rendered  immune  is  capable  of  neutralizing  all  effects  of  the  pollen  toxin. 

As  regards  the  standardization  of  this  serum,  it  is  effected  by  mixing 
the  dosis  minima  certe  efficax  of  the  toxin  with  various  dilutions  of  the 
serum  and  instilling  the  mixture  into  the  conjunctival  sac  of  individuals 
with  a  tendency  for  hay-fever.  That  amount  of  the  serum  which  suffices 
to  neutralize  the  toxic  action  is  taken  as  the  unit  of  measure.  Sera  of  at 
least  thirty  times  the  unit  strength  are  selected  for  therapeutic  application. 

The  immune  serum  is  manufactured  in  fluid  and  powder  form,  and  is 
placed  on  the  market  under  the  name  of  "Pollantin."  Its  use  is  mainly 
local,  by  spraying  a  small  quantity  of  the  pollen  powder  upon  the  nasal 
mucosa  several  times  daily  and  by  placing  several  granules  into  the 
conjunctival  sac  with  a  cameFs-hair  brush.  The  serum  can  also  be  em- 
ployed as  a  prophylactic. 

If  the  eyes  are  especially  reddened,  it  is  best  to  deposit  some  fluid  serum 
into  the  conjunctival  sac  every  day.  Prausnitz  advises  the  injection  of  i 
to  2  c.cm.  of  the  serum  subcutaneously  when  asthmatic  attacks  occur, 
when  the  above  local  treatment  has  failed. 

In  America  a  special  Pollantin  is  made  against  the  frequent  form  of 
hay-fever  known  as  "autumn  catarrh"  by  immunization  with  the  pollen 
of  the  golden  rod  and  rag  weed. 

The  pollantin  therapy  and  prophylaxis  has  been  quite  satisfactory, 
inasmuch  as  two-thirds  of  the  patients  remain  either  entirely  free  from 
attacks  or  are  so  greatly  benefited  that  their  general  duties  are  not  inter- 
fered with. 

The  specificity  of  anaphylaxis  has  been  proposed  for  diagnostic  aid. 
As  yet  the  methods  have  not  been  perfected  sufficiently  to  be  of  clinical 
value. 


CHAPTER  XVIII. 

PASSIVE  IMMUNIZATION  (SERUM  THERAPY).     BACTERIOLYTIC  SERA. 
SPECIAL  SERUM  THERAPY. 

In  the  former  chapters  it  was  proven  that  during  active  immunization 
of  an  animal  specific  protective  bodies  were  formed  which  circulate  in  the 
blood  and  can  by  means  of  the  serum  be  transferred  to  another  organism. 
It  was  further  found  that  such  bodies  exert  this  protection  against  fatal 
intoxication  or  infection  in  various  ways;  thus,  as  antitoxins  and  anti- 
aggressins  they  neutralize  toxic  poisons  and  aggressins;  as  bacteriolysins 
they  bring  about  lysis  of  the  bacteria;  while  as  bacteriotropins  they  prepare 
the  bacteria  for  phagocytosis.  The  defensive  qualities  of  such  a  transferred 
serum  are  evident  not  only  if  the  infection  is  incited  at  the  same  time  as,  or 
a  short  period  after  the  serum  is  given,  but  in  numerous  instances  curative 
effects  are  observed  if  the  serum  is  given  even  after  infection  has  already 
taken  place. 

Of  all  sera,  those  with  antitoxic  properties  have  met  the  greatest  suc- 
cess in  therapeutic  application.  They  have  already  been  referred  to  in 
their  respective  chapters. 

The  efficiency  of  the  pure  bacteriolytic  sera  on  the  other  hand  has  been  dis- 
appointing. One  reason  given  for  this  lack  of  curative  action  is,  the  in- 
ability of  bacteriolytic  serum  to  neutralize  the  endotoxins. 

Pfeiffer's  experiment  revealed  that  if  the  number  of  bacteria  exceeded  a  certain 
limit,  then  in  spite  of  bacteriolysis,  death  of  the  animal  takes  place.  This  was  explained 
by  the  existence  of  endotoxins.  By  bacteriolysis  the  endotoxins  normally  confined 
within  the  bacteria  are  liberated  and  thus  get  a  chance  to  show  their  toxicity. 

The  aim,  therefore,  was  to  produce  antiendotoxic  sera.  The  accomplishment  of 
this  was  prevented  by  the  erroneous  view  of  Wolff-Eisner  who  claimed  that  it  was 
impossible  to  immunize  against  endotoxin. 

Numerous  methods  have  been  advocated  for  the  liberation  of  these  endotoxins: 
maceration  of  bacteria,  exposure  to  very  low  temperature,  admixture  with  chemical 
substances  which  would  dissolve  the  outer  capsule,  ferment  digestion,  growth  upon 
certain  culture  media,  etc.  At  the  present  day,  there  is  absolutely  no  doubt  that  the 
bacterial  bodies  contain  poisonous  substances  against  which  it  is  difficult  and  to  a  cer- 
tain degree  impossible  to  attain  an  immunity. 

Whether  one  should  adhere  to  the  old  idea  and  apply  to  these  the  term  endotoxin, 
or  include  them  in  the  class  of  true  toxins  with  the  only  difference  that  they  are  not 
secreted  but  contained  within  the  bacterial  body  and  therefore  more  difficult  to  isolate, 
is  purely  a  question  of  theoretical  importance. 

Another  cause  for  the  therapeutic  failure  of  bacteriolytic  serum,  as  ad- 

231 


232  PASSIVE   IMMUNIZATION 

vocated  by  Bail  and  his  school,  is  the  lack  of  its  antiaggressin  action.  This 
applies  only  to  the  cases  in  which  the  bacteriolytic  serum  was  produced  by 
immunization  with  dead  bacteria. 

When  live  bacteria  are  used,  this  objection  is  not  to  be  considered,  as 
according  to  the  experiments  of  Wassermann  and  Citron,  "aggressin"  is 
nothing  more  than  the  immunizing  substance  of  the  living  bacteria.  As 
far  as  the  structure  of  the  antiaggressins  is  concerned,  the  author  was 
able  to  show  that  like  the  bacteriolysins,  they  are  amboceptors  which 
bind  complement. 

Artificial  aqueous  extracts  of  living  bacteria,  belonging  to  the  class  of 
half  parasites  made  according  to  the  method  of  Wassermann  and  Citron, 
contain  the  endotoxin  as  well  as  the  aggressin.  Such  artificial  aggressins, 
therefore,  represent  ideal  antigens.  The  sera  produced  by  their  injection 
contain  but  few  bacteriolytic  bodies  and  a  very  large  number  of  ambo- 
ceptors, easily  demonstrable  by  the  Bordet-Gengoa  reaction. 

Wassermann  explains  the  lack  of  therapeutic  efficiency  on  the  part  of 
the  bacteriolytic  sera  by  the  absence  of  complement  of  the  organisms,  as 
well  as  by  the  inability  of  human  complement  invariably  to  fit  animal 
amboceptors.  As  is  known,  amboceptors  increase  during  immunization 
while  the  complement  content  remains  the  same.  But  since  amboceptors 
without  complement  remain  inactive,  even  a  very  strong  serum  may  only 
be  slightly  effective,  depending  upon  the  amount  of  existing  complement. 
If  too  many  amboceptors  are  injected,  the  serum  may  become  entirely 
powerless  due  to  a  phenomenon  similar  to  Neisser  and  Wechsberg's 
complement  deviation.  Wassermann  advises  therefore  the  addition  of 
complement  to  a  serum  before  its  injection,  in  order  to  activate  it.  This 
suggestion  has  not  been  widely  adopted  in  practice. 

It  is  for  a  similar  reason,  that  the  classical  experiment  of  bacteriolysis 
is  so  beautifully  demonstrable  in  the  guinea-pig's  peritoneal  cavity,  an 
area  relatively  poor  in  cells,  while  this  phenomenon  is  incomplete  and 
replaced  by  phagocytosis  when  occurring  in  the  blood,  inner  organs,  and 
subcutaneous  connective  tissue.  It  is  in  this  connection  that  Metch- 
nikoff  and  his  followers  see  the  main  reason  for  the  failure  of  the  therapeutic 
activity  of  bacteriolytic  sera. 

An  additional  impediment  is  offered  by  the  wide  differences  which 
exist  among  the  numerous  strains  of  the  same  bacterium.  This  may  be 
so  marked  that  an  immune  serum  produced  with  one  strain  will  offer  no 
protection  against  a  different  strain  of  the  same  bacterium.  It  is  now 
overcome  to  a  certain  extent  by  immunization  with  as  many  different 
strains  of  the  same  bacterium  as  possible  (polyvalent  sera). 

Cultures  grown  upon  artificial  media  for  a  very  long  time  adapt  them- 
selves to  their  new  surroundings  and  frequently  lose  some  of  their  bio- 
logical characteristics,  e.g.,  virulence.  If  the  culture  is  then  inoculated 


POSSIBILITIES    OF    SERUM   THERAPY  233 

into  an  animal,  the  virulence  is  increased  usually  only  for  that  animal 
species,  but  may  remain  the  same  or  even  lowered  for  man.  Many 
authors,  therefore,  employ  for  the  production  of  immune  sera  only  virulent 
strains  of  bacteria  freshly  isolated  from  a  human  being. 

In  spite  of  all  the  above  considerations,  the  fact  still  remains  that  most 
immune  sera  excepting  those  of  the  cholera,  typhoid,  and  paratyphoid 
bacteria,  show  no  bacteriolytic  tendencies  even  under  the  most  favorable 
circumstances;  but  by  means  of  their  amboceptors  they  fix  free  comple- 
ment and  with  the  aid  of  bacteriotropins,  stimulate  phagocytosis. 

Whether  complement  fixation  is  at  all  to  be  considered  as  a  protective 
phenomenon,  cannot  with  the  evidence  existing  at  present  be  definitely 
decided. 

Conditions  are  much  more  favorable  as  far  as  the  bacteriotropins  are 
concerned.  Active  phagocytosis  is  always  an  expression  of  good  resistance. 
It  is  not  necessary  for  the  leucocytes  to  digest  the  bacteria;  it  is 
amply  sufficient  if  a  protective  wall  of  these  cells  is  formed  (Ribbert, 
Citron,  Gruber) ;  moreover  they  can  neutralize  the  bacterial  poisons.  In 
this  connection  it  must  always  be  borne  in  mind  that  phagocytosis  by  no 
means  necessitates  the  death  of  bacteria. 

Granting,  however,  that  all  the  above  requirements  have  been  fulfilled 
and  a  suitable  serum  has  actually  been  produced,  will  such  a  serum  always 
be  effective,  or  are  there  any  other  causes  which  may  interfere  with  its 
good  results?  In  order  to  answer  this,  the  infectious  diseases  must  be 
divided  into  acute  and  chronic.  With  the  first  class,  success  is  quite 
assured  as  long  as  it  is  possible  to  bring  sufficient  amounts  of  the  active 
serum  substances  into  direct  contact  with  the  bacteria.  In  meningeal 
infections,  subdural  injections  may  have  to  be  adopted.  It  is  difficult, 
however,  in  cases  of  this  nature  to  judge  definitely  whether  the  serum 
therapy  was  really  the  effective  agent,  inasmuch  as  diseases  like  erysipelas, 
meningitis,  pneumonia,  etc.,  are  self  limited,  lasting  for  a  period  of  time 
and  then  subsiding  of  their  own  accord. 

With  the  chronic  infections,  on  the  other  hand  (especially  tuberculosis), 
serum  therapy  has  a  new  difficulty  to  overcome.  As  a  result  of  the  long 
course  of  the  disease,  it  is  naturally  impossible  by  means  of  a  single 
injection  to  introduce  sufficient  curative  bodies,  as  can  be  done  in  diph- 
theria, for  example.  It  is  necessary,  therefore,  frequently  to  repeat  the 
injections.  Under  such  conditions  the  human  organism  produces  anti- 
bodies against  the  foreign  proteid  (anaphylaxis) ,  perhaps  even  against  the 
curative  substances  in  the  serum  (antiamboceptors) .  In  both  instances 
the  desired  effect  of  the  serum  is  lost.  A  further  impediment  lies  in 
the  possibility  that  bacteria  remaining  in  a  system  for  a  long  period 
immunize  themselves,  and  thus  resist  the  action  of  the  antibodies  directed 
against  them.  Such  bacterial  strains  are  known  as  "  serum  fast." 


234  PASSIVE  IMMUNIZATION 

Special  Serum  Therapy. 

i.  Meningococcus  Serum. — Numerous  investigators  have  attempted  the 

Meningococ-  production  of  an  immune  serum  for  man,  among  these  Jochmann,  the 

cus  Immune  Berlin  Institute  for  infectious  diseases,  Ruppel,  Kraus,  Flexner  and 

Serum.      Jobling,   and   others.     The   sera  of  Jochmann   (Merck)    and  Ruppel 

(Hochst)  are  produced  by  immunization  of  horses  with  meningococci 
which  are  at  first  employed  in  dead,  and  later  in  live  form.  The  other  sera  mentioned 
are  obtained  by  immunization  with  bacterial  extracts  or  bacterial  extracts  plus  full 
bacteria,  and  therefore  contain  agglutinins,  precipitins,  bacteriotropins,  amboceptors 
and  antiendotoxins.  It  is  difficult  to  test  the  efficiency  of  these  sera  in  animals,  as  the 
meningococci  vary  greatly  in  their  virulence  toward  them.  Jochmann  and  Ruppel 
assert  that  they  have  been  successful  in  growing  cultures  extremely  virulent  for  ani- 
mals which  they  employed  for  the  titration  of  the  therapeutic  value  of  the  serum.  In 
the  institute  for  infectious  diseases,  the  method  of  complement  fixation  is  taken  as 
the  index  of  the  therapeutic  value  of  the  serum.  This  procedure  is  very  unreliable. 
The  protection  of  the  serum  in  mice  against  the  meningococcus  endotoxin  as  well  as 
the  demonstration  of  the  bacteriotropic  action  of  the  serum  is  far  more  significant. 

In  man,  the  immune  serum  is  injected  subdurally,  after  a  quantity 
of  spinal  fluid  has  been  withdrawn  to  relieve  the  pressure.  In  adults  20  to 
40  c.cm.  and  in  children  10  to  20  c.cm.  are  daily  injected  until  there  is 
clinical  improvement  or  a  fatal  prognosis  becomes  inevitable.  It  is  ad- 
visable to  precede  the  serum  inoculation  by  a  morphine  injection,  and  to 
elevate  the  pelvis  for  eight  to  twelve  hours  after  the  inoculation.  The 
earlier  the  serum  therapy  is  instituted,  the  more  favorable  are  its  results. 
Subcutaneous  applications  of  the  serum  or  employment  of  a  serum  more 
than  three  months  old  is  absolutely  of  no  use. 

Both  in  the  United  States  and  in  foreign  countries  the  value  of  the  serum  as  a  thera- 
peutic agent  seems  fairly  established.  In  Germany,  the  serum  is  obtained  gratis  at 
the  institute  for  infectious  diseases  at  Berlin.  The  serum  in  Switzerland  is  distributed 
by  the  serum  institute  of  Bern  (Kolle).  In  the  United  States,  the  Rockefeller  Institute 
of  New  York  first  conducted  its  dispensation,  but  now  it  is  under  the  supervision  of  the 
New  York  Board  of  Health. 

Numerous  statistics  can  be  cited  exemplifying  the  good  results  of  the  serum.  The 
following  figures  given  by  Levy  describing  the  experiences  in  the  Essen  epidemic  are 
especially  instructive : 

From  the  first  of  January  until  the  first  of  November,  1907,  the  total  number  of 
epidemic  meningitis  cases  which  occurred  in  Essen  were: 

55  cases  with  29  deaths  =  5 2.72%  mortality, 
of  these,  treatment  was  given  outside  of  the  barracks  to 

15  cases  with  12  deaths  =  80%  mortality, 
inside  the  barracks  were  treated 

40  cases  with  17  deaths =4 2. 5%  mortality, 
of  these 

14  cases  were  not  treated  with  serum  with  n  deaths  as  a  result 
=  78.6%  mortality, 


SERUM   THERAPY   IN   CEREBRO SPINAL   MENINGITIS 


235 


those  treated  with  serum  were 

23  cases  with  5  deaths  =2 1.7%  mortality, 

of  these,  those  which  were  treated  only  incompletely  (subcutaneously)  and  with  insuffi- 
cient doses,  numbered 

6  cases  with  3  deaths  as  the  outcome  =50%  mortality, 
systematic  subdural  treatment  with  large  doses. 

17  cases  with  2  (i)  deaths=n.8  (6.3)%  mortality. 

The  figures  in  parentheses  represent  the  moribund  cases  coming  under  treatment  and 
the  percentage  which  would  result  if  these  were  not  included  in  the  calculation. 

The  experiences  with  the  serum  of  Flexner  and  Jobling  are  similarly  encouraging. 
In  a  report  of  400  cases  (1909)  the  mortality  is  reported  as  lowered  from  80  per  cent, 
to  20  per  cent. 


In  a  more  recent  report  (1913),  1,294  patients  treated  with  serum  show 
the  following  results:  recovered  894;  died  400  (30.9  per  cent.).  Flexner's 
tables  which  are  here  reproduced  are  instructive. 


MORTALITY  ACCORDING   TO   THE   PERIOD   OF   INJECTION   OF  THE 

SERUM. 


. 

Period  of  Injection 

No.  of  cases 

Recovered 

Died 

Per  cent, 
recovered 

Per  cent, 
died 

ist  to  3d  day  

100 

163 

36 

81.9 

18.1 

4th  to  7th  day  

346 

252 

94 

72.8 

27.2 

Later  than  7th  day.  .  . 

666 

423 

243 

63.5 

36.5 

Totals.   . 

I   211 

8^8 

•27-7 

60.2 

30.8 

030 

MORTALITY  ACCORDING  TO  AGE. 


Age 

No.  of  cases 

Recovered 

Died 

Per  cent, 
recovered 

Per  cent. 

died 

Under'    i  year 

1  20 

6s 

64. 

SO  4 

49.  6 

i     to    2  years  
2    to    5  years  
5    to  10  years  
10    to  20  years.  .  .   . 

87 
194 
218 
360 

60 
i39 
185 

2C4 

27 
55 
33 
106 

69.0 
71.6 
84.9 
70.  6 

31.0 
28.4 

i5-i 
29.4 

Over     20  years  
Age  not  given  

288 
18 

180 
ii 

108 

7 

62.5 
61.1 

37.5 

38.9 

Totals  

I   2Q4. 

804. 

400 

.  .• 
69.1 

30.9 

236  PASSIVE  IMMUNIZATION 

MORTALITY  ACCORDING  TO  AGE  AND  PERIOD  OF  INJECTION. 


Injected  ist  to 

Injected  4th  to 

Injected  later  than 

3d  day 

7th  day 

7th  day 

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Age 

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Totals  .  . 

199 

163 

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343 

250 

93 

27.1 

664 

421 

243    36.6 

2.  Streptococcus  Immune  Sera. — The  role  of  the  streptococcus  in  some  diseases,  for 
example,  scarlet  fever,  is  imperfectly  understood.  Moreover  it  has  only  been  in- 
definitely established  whether  there  are  various  groups  or  only  one  kind  of  strepto- 
coccus; even  the  significance  of  their  virulence  or  hemolysin  formation  is  not  clear. 
These  difficulties  account  for  the  great  number  of  methods  advocated  for  the  pro- 
duction of  an  immune  streptococcus  serum.  The  oldest,  serum  is  that  of  Marmorek. 
It  was  produced  by  immunization  with  a  strain  made  highly  virulent  by  passage 
through  animals.  The  other  sera  on  the  market  are: 

a.  Serum  of  Aronson  (Schering). — This  is  a  polyvalent  serum  produced  by  immuni- 
zation of  horses  with  cultures  pathogenic  for  man;  some  strains  having  previously 
been  passed  through  animals,  others  not.     The  strength  of  the  serum  is  tested  in  mice 
infected  with  the  latter  strains. 

b.  Serum  of  Meyer-Ruppel  (Hochst  Farbwerke). — Horses  are  first  immunized  with  a 
strain  of  streptococcus  whose  virulence  has  been  raised  by  passage  through  horses  and 
mice;  each  horse  is  then  injected  with  a  different  strain  of  human  streptococcus.     When 
the  serum  of  each  animal  is  of  such  a  strength  that  doses  of  o.oi  to  0.0005  c.cm.  protect 
mice  infected  with  its  own  particular  strain,  the  sera  of  the  different  horses  are  mixed. 
Thus  a  polyvalent  serum  is  obtained. 

c.  Serum  of  Menzer  (Merck)  is  monovalent  and  produced  by  immunization  with  a 
culture  which  is  pathogenic  for  man  and  not  passed  through  animals. 

d.  Serum  of  Moser  is  polyvalent,  produced   by  injections  of    streptococci  from 
scarlet  fever.    The  sera  of  Menzer  and  Moser  are  not  tested  by  injections  of  white  mice. 
The  others  are.     One  cannot  strictly  rely  upon  this  method  of  serum  titration  for  its 
employment  in  man.     The  virulence  of  streptococci  against  mice  and  human  beings 
bears  no  definite  relation.     A  serum  may  be  perfectly  efficient  in  mice  both  for  prophy- 
lactic and  therapeutic  purposes,  and  be  entirely  inactive  in  man;  also  vice  versa.     The 
action  of  the  serum  should  be  in  the  main  of  bacteriotropic  nature. 

Antistreptococcus  serum  has  been  tried  in  scarlet  fever,  puerperal  sep- 
sis, erysipelas,  and  articular  rheumatism. 

Complement  fixation   experiments    (Foix   and   Mallein,    Schleissner) 


STREPTOCOCCUS   AND   PNEUMOCOCCUS    IMMUNE    SERA  237 

have  shown  that  the  streptococci  of  scarlet  fever  can  be  definitely  sepa- 
rated from  the  other  varieties  of  these  bacteria.  This  work  could  not 
be  confirmed. 

In  this  disease  favorable  results  have  been  observed  by  the  use  of 
Moser's  serum. 

Escherich  states  that  of  112  scarlet  fever  cases  injected,  those  receiving  the  serum 
on  the  first  and  second  days  of  their  illness  all  recovered,  while  of  those  injected  later 
on,  there  was  a  high  percentage  of  mortality.  Other  authorities  have  seen  no,  or  only 
very  slight  effect  from  the  serum  treatment. 

Two  hundred  cubic  centimeters  of  Moser's  serum  must  be  given  subcutaneously. 

The  treatment  of  puerperal  fever  has  been  favorably  influenced  by 
Aronson's  and  Meyer-Ruppel's  serum,  of  which  50  c.cm.  are  injected  on 
several  successive  days. 

Menzer's  serum  is  said  to  serve  its  purpose  best  in  acute  and  chronic 
rheumatism  as  well  as  in  tuberculous  mixed  infections. 

In  erysipelas  all  the  well  known  sera  have  been  tried.  On  account  of  the  very 
variable  course  of  the  disease  it  is  difficult  to  judge  the  exact  value  of  the  serum. 
In  fact,  thus  far  one  cannot  with  certainty  depend  upon  any  serum  treatment  of  a 
streptococcus  infection,  but  the  serious  nature  of  such  infections  makes  every  possible 
therapeutic  measure  strongly  justifiable. 

3.  The  pneumococcic  sera  most  frequently  used  are  those  of  Pane, 
Pneumococ-   Romer  and  Merck.     Pane  immunizes  donkeys  with  highly  virulent  pneu- 
cus  Immune  mococci  and  uses  the  serum  for  the  treatment  of  pneumonia.     Several 
Sera.        Italian  investigators  record  favorable  results. 

Romer  prepares  a  polyvalent  serum  by  injecting  horses  with  different 
strains  of  pneumococcus  obtained  directly  from  man;  the  strength  of  the  serum  is  tested 
in  mice.  The  serum  is  mainly  employed  both  for  the  protection  and  cure  of  ulcus 
cornea  serpens. 

The  result  according  to  Romer  depends  upon  the  very  variable  virulence  of  the 
pneumococci.  The  severity  of  the  infection  in  man  is  said  to  run  parallel  with  the 
virulence  in  mice.  Romer,  therefore,  ascertains  in  every  case  of  ulcus  serpens  whether 
his  serum  has  any  protective  bodies  for  that  particular  strain  of  pneumococcus,  and 
tests  the  virulence  of  the  same. 

The  serum  can  be  injected  intravenously  and  subcutaneously,  and  in  pneumococcus 
meningitis,  subdurally.  It  is  manufactured  by  the  Hochst  Farbwerke,  in  vials  of  10 
and  20  c.cm. 

A  similar  serum  is  manufactured  by  Merck.  It  is  obtained  from  horses  and  stand- 
ardized at  the  Institute  for  experimental  therapy  in  Frankfort  so  that  o.oi  c.cm. 
injected  subcutaneously  protects  a  mouse  inoculated  intraperitoneally  24  hours  later 
with  10  to  100  times  the  lethal  dose  of  a  living  pneumococcus  culture.  This  is 
known  as  a  normal  serum  and  i  c.cm.  contains  one  immunity  unit  (I.  E.).  The  serum 
on  the  market  contains  20  to  40  units  per  c.cm. 

In  pneumonia  200  to  400  units  are  given  subcutaneously  and  repeated  in  three  to 
four  days,  if  the  fever  does  not  subside.  As  a  prophylactic  inoculation,  200  to  400 
units  are  given  to  old  people  where  a  "hypostatic"  pneumonia  is  feared.  In  ulcus 
serpens  of  the  cornea  200  to  400  units  are  employed  and  if  no  improvement  sets  in,  the 
dose  is  repeated  upon  the  third  day.  In  addition,  several  drops  of  the  serum  are 


238  PASSIVE  IMMUNIZATION 

instilled  into  the  conjunctival  sac  every  two  hours.     As  a  prophylactic  dose  in  this 
disease,  100  units  suffice. 

Merck  also  prepares  a  vaccine  of  dead  pneumococci  in  doses  of  i  c.cm.  which  further 
aid  in  the  treatment  of  pneumococci  infections.  One  cubic  centimeter  of  such  dead 
pneumococci  can  be  administered  for  the  prophylaxis  of  ulcus  serpens. 

4.  Pest  Sera. — A  large  number  of  pest  sera  are  in  use. 

a.  The  Paris  serum  (Yersin)  produced  at  Pasteur  Institute  by  immunization  of 
horses  with  dead  and  later  on  living  bacilli. 

b.  The  Bern  serum  of  Tavel  employs  the  same  principles. 

c.  Lustig's  Serum. — For  this  serum,  horses  are  immunized  with  the  pest-nucleo- 
proteids.     Pest  cultures  are  broken  up  by  i  per  cent,  of  potassium  hydroxide  and  from 
this,  by  the  addition  of  acetic  acid,  the  nucleoproteid  is  precipitated  and  then  suspended 
in  salt  solution  to  serve  as  antigen. 

d.  Serum  of  Terni-Bandi  is  prepared  by  the  immunization  of  donkeys  and  sheep 
with  natural  pest  aggressins. 

e.  Serum  of  Markl  is  supposedly  an  antitoxic  serum  prepared  by  immunization 
with  nitrates  of  old  pest  bouillon  cultures. 

All  the  above  sera  contain  agglutinins,  precipitins,  bacteriotropins  and  ambocep- 
tors;  the  serum  of  Terni-Bandi  contains  aggressin  amboceptors,  that  of  Markl,  anti- 
endotoxins. 

The  sera  are  tested  for  their  anti-infectious  properties  in  animals  such  as  guinea- 
pigs,  rats,  mice.  Markl  also  estimates  the  toxin  neutralization  power  of  his  serum. 

The  Paris  serum  comes  either  in  dry  form  or  in  bottles  containing  20  c.cm.  without 
any  preservatives.  Ten  to  20  c.cm.  should  suffice  as  a  prophylactic  injection,  although 
Martini  advises  100  c.cm.  at  least.  The  period  of  protection  is  short,  averaging  about 
fourteen  days. 

Prophylactic  injection  is  advisable  in  those  instances  where  an  immediate  protection 
is  necessary,  like  the  inoculation  of  physicians  and  nurses  attending  pest  patients. 
Under  all  other  circumstances  either  active  immunization  or  the  simultaneous  method 
of  Shiga  should  receive  the  preference. 

For  the  treatment  of  pest  infections,  Calmette  and  Salimbeni  advise  intravenous 
administration  of  20  c.cm.  and  two  subcutaneous  injections  of  40  c.cm.  each — all  to  be 
given  on  the  first  day;  on  the  second  day  two  similar  subcutaneous  injections;  and  if 
the  case  is  of  a  severe  nature,  the  dose  may  be  doubled.  The  results  are  variable. 

From  comparative  studies,  it  seems  that  Lustig's  serum  is  somewhat  weaker  than 
the  Paris  serum.  The  sera  of  Terni-Bandi  and  Markl  have  not  been  sufficiently 
employed,  so  that  opinion  is  reserved. 

5.  Tuberculosis  Sera. — The  best  known  and  most  studied  are  those  of  Maragliano 
and  Jarmorek. 

a.  Serum  of  Maragliano  is  prepared  by  Maragliano's  institute  in  Genoa  from  horses 
which  are  immunized  for  about  six  months  with  the  soluble  substances  of  tubercle  ba- 
cilli.    The  favorable  action  of  the  serum  is  reported  on,  especially  by  Italian  authorities. 

b.  Serum  of  Marmorek  is  prepared  in  the  laboratory  of  Marmorek,  at  Paris-Neuilly, 
by  the  immunization  of  horses  with  the  so-called  "primitive"  tubercle  bacilli,  i.e., 
young  tubercle  bacilli  whose  acid-fast  character  is  still  very  slight  or  entirely  absent. 
When  the  horses  have  attained  a  high  grade  of  immunity,  they  receive  injections  of 
various  strains  of  pure  cultures  of  streptococci  obtained  from  the  sputum  of  tubercu- 
lous patients.     The   serum  of  these  animals  is,  therefore,  antituberculous  and  at  the 
same  time  polyvalent  antistreptococcic  (a  double  serum),  serving  against  the  mixed 
infections. 

This  serum  is  administered  daily,  either  subcutaneously  5  to  10  c.cm.  or  per  rectum 


SPECIFIC    SERUM   THERAPY  239 

20  c.cm.  The  latter  form  is  more  advisable  for  the  sake  of  preventing  anaphylaxis. 
Citron  has  found  the  serum  entirely  harmless,  the  bad  effects  described  by  some  being 
probably  due  to  the  idiosyncrasy  of  patients  against  foreign  sera.  The  most  favorable 
results  have  been  claimed  in  localized  bone  and  joint  tuberculosis  and  in  the  incipient 
stages  of  pulmonary  tuberculosis.  Especial  consideration  of  the  serum  should  be  given 
in  those  patients  who  evince  persistent  temperature  or  the  very  severe  but  not  hopeless 
cases,  where  the  tuberculin  therapy  cannot  be  undertaken.  In  some  of  these  instances 
very  encouraging  results  have  been  noted. 

Occasionally  the  author  started  with  the  serum  treatment,  and  then  combined  with 
it  the  tuberculin  administration  and  finally  left  the  serum  entirely. 

6.  Anthrax  Sera. — Sclavo,  Deutsh,  Sobernheim  and  others  have  produced  immune 
sera  by  the  immunization  of  donkeys,  sheep  and  horses.     These  have  been  mainly 
employed  in  veterinary  practice. 

In  man  the  serum  has  been  tried  only  by  Sclavo.  He  injects  30  to  40  c.cm.  sub- 
cutaneously  for  several  successive  days;  in  severe  infections  10  c.cm.  are  administered 
intravenously.  Two  cases  described  by  Bandi  received  150  c.cm.  intravenously. 

7.  Typhoid  Immune  Sera. — The  ordinary  bacteriolytic  sera  (Tavel)  have  not  met 
with  the  desired  success  in  the  therapy  of  typhoid  fever.     Attempts  have,  therefore, 
been  made  to  produce  antiendotoxic  sera.     Chantemesse  treats  horses  for  several 
years  with  bouillon  nitrates;  Besredka  injects  first  dead  and  then  living  typhoid  bac- 
teria from  agar  cultures,  Mac  Fadyen  breaks  up  the  bacteria  at  very  low  temperatures 
and  thus  liberates  the  endotoxin  for  purposes  of  immunization.     Kraus  and  von  Sten- 
itzer  use  bouillon  filtrates  and  aqueous  bacterial  extracts  as  is  likewise  done  by  Meyer- 
Bergell  and  Aronson.     Garbat  and  Meyer  employ  sensitized  typhoid  bacilli,  i.e., 
bacteria  united  with  their  bacteriolytic  amboceptors. 

Chantemesse  injects  several  drops  of  his  serum  subcutaneously.  Its  effect  lasts 
ten  days.  Only  occasionally  is  a  second  inoculation  necessary;  if  so,  it  must  be  much 
smaller.  His  results  have  been  good  and  have  mainly  depended  upon  an  increase  in 
the  opsonic  index. 

Meyer  and  Bergell  as  well  as  Kraus  give  20  to  50  c.cm.  subcutaneously. 

8.  Cholera  Serum.- — Similar  attempts  for  the  production  of  a  cholera  antiendotoxic 
serum  have  been  made.    "Kraus  has  succeeded  in  obtaining  an  antitoxin  against  some 
El-Tor  vibrios  which  have  all  the  characteristics  of  true  cholera  vibrios. 

The  experiments  with  Kraus'  serum,  and  Kolle's  serum  (Bern  Institute),  at  present 
being  conducted  in  Russia,  seem  to  be  favorable. 

The  serum  therapy  of  infectious  diseases  is  still  in  its  prime.  The 
contradictory  results  of  many  authors  are  to  be  attributed  not  only  to 
the  variable  efficiency  of  the  sera,  but  also  to  the  method,  the  time,  and 
the  dose  chosen  for  administration. 

The  same  serum  in  the  hands  of  different  physicians  may  yield  opposite 
results.  These  subjective  sources  of  error  must  be  overcome  or  minimized 
by  making  a  complete  and  thorough  study  of  the  effects  which  a  certain 
serum  may  have  and  actually  does  have;  here  all  the  clinical  and  laboratory 
guides  must  be  made  use  of.  Employed  in  this  manner,  serum  therapy 
will  even  at  the  present  stage  lead  to  beneficial  results. 

Wright's  motto  at  the  beginning  of  his  book  on  vaccines  "The  physi- 
cian of  the  future  will  be  an  immunizator,"  can  justly  be  reversed  to  read, 
"the  immunizator  of  the  future  will  be  the  physician  in  the  true  sense." 


CHAPTER  XIX. 

CHEMOTHERAPY. 

DEFINITION.     METHOD.    ATOXYL.    SALVARSAN.     CHEMOTHERAPY    or    MALIGNANT 

TUMORS.     CONCLUSION. 

Serum  therapy  proved  the  fundamental  fact  that  it  is  possible  by 
the  injection  of  specific  sera  selectively  to  destroy  or  counteract  the 
poisonous  effects  of  certain  micro-organisms  without  in  any  way  injuring 
the  infected  host.  That  similar  results  are  attainable  by  chemical  means 
is  demonstrated  by  the  empirical  use  of  quinine  in  malaria.  For  a  long 
time  this  chemical  specific  stood  in  a  class  by  itself.  In  recent  years, 
the  progress  made  in  the  study  of  infections  by  the  protozoa,  especially 
the  trypanosomes  and  spirochetes,  and  the  possibility  of  transmitting 
these  diseases  to  the  lower  animals,  stimulated  a  renewed  effort  in  quest 
of  chemical  substances  analogous  to  quinine.  Paul  Ehrlich  led  the  way 
in  this  new  direction  and  termed  this  study  "  Chemotherapy "  in  contra- 
distinction to  "Pharmacotherapy." 

Only  those  agents  can  be  employed  chemotherapeutically  in  which  the 
"  organatrope"  and  "parasitatrope"  relation  is  favorable,  that  is,  primarily 
when  the  curative  dose  is  only  a  very  small  fraction  of  the  toxic  dose 
(Ehrlich).  In  infectious  diseases  it  is  the  causative  parasite  that  is 
aimed  at,  in  malignant  tumors  the  tumor  cells  are  the  objects  for  de- 
struction. In  both,  however,  it  is  absolutely  essential  that  the  normal 
tissues  of  the  body  remain  entirely  uninjured. 

Thus  far  the  most  favorable  results  of  chemotherapy  have  been  ac- 
complished in  trypanosome  and  spirochete  infections. 

Researches  under  Ehrlich's  direction  clearly  defined  that  there  are  three  different 
types  of  substances  which  can  destroy  the  trypanosomes : 

(a)  The  group  of  basic  silk  dyes  (Fuchsin). 

(b)  The  group  of  cotton  dyes  (Benzopurpurin  series)  of  which  trypan  red  and  try- 
pan  blue  have  proved  most  efficient. 

(c)  The  group  of  arsenical  products  (atoxyl  and  its  derivatives). 

The  basis  for  differentiating  these  three  classes  is  seen  from  the 
following  experiments.  If  a  mouse  suffering  from  trypanosomiasis 
receives  an  injection  of  an  active  fuchsin  preparation,  the  trypanosomes 
disappear  from  the  blood.  They  remain  away  permanently  ("  Sterilisatio 
magna")  provided  that  the  injected  dose  is  large  enough.  The  animal 
is  thus  cured  by  a  single  injection.  If  the  dose  is  not  sufficient,  many 

240 


PRINCIPLES    OF   CHEMOTHERAPY  241 

of  the  parasites  are  destroyed,  but  some  remain  alive.  This  number 
may  be  so  small  that  for  several  days  the  blood  may  seem  sterile,  but 
then  the  remaining  trypanosomes  multiply  and  a  relapse  occurs.  The 
same  fuchsin  preparation  is  again  injected  and  now,  one  may  attain 
a  complete  cure  if  the  dose  this  time  is  sufficiently  large;  but  if  not  a 
relapse  sets  in. 

Thus  the  treatment  is  continued.  If,  however,  the  relapses  are 
numerous  and  the  same  curative  agent  is  repeatedly  employed,  a 
state  is  reached  in  which  the  fuchsin  no  longer  has  any  beneficial  influence 
whatever.  The  trypanosomes  have  become  immunized  or  acclimated  to 
fuchsin,  in  other  words,  have  become  "fuchsin-fast." 

To  prove  that  it  is  the  trypanosomes  that  have  acquired  a  new  characteristic  and 
not  that  the  organism  of  the  mouse  had  become  altered  in  its  susceptibility,  one  may 
infect  for  the  first  time  another  mouse  with  this  "  f uchsin-f ast "  strain,  and  here  again 
this  dye  will  have  no  effect  at  all.  The  artificially  attained  resistance  toward  fuchsin 
remains  as  a  permanent  characteristic  of  this  particular  strain  of  trypanosomes,  even 
though  transplanted  from  mouse  to  mouse  for  years.  It  disappears,  however,  if 
the  parasites  are  allowed  to  increase  by  a  sexual  cycle  of  development  which  is 
possible  for  example  in  the  rat  louse  Hematopinus  spinolosus. 

This  fuchsin-fast  character  has  not  at  the  same  time  altered  the 
susceptibility  of  the  trypanosomes  toward  any  of  the  trypanocidal 
agents  of  the  second  or  third  group  as  trypan  red  or  atoxyl.  Just  as 
guinea-pigs  immunized  against  cholera  may  succumb  to  infection  by  the 
typhoid  bacillus,  so  also  may  trypanosomes,  which  have  become  inert  to 
fuchsin,  be  attacked  by  trypan  red  or  atoxyl.  Furthermore  the  acquired 
resistance  of  the  protozoa  to  the  action  of  these  various  chemicals  is  just  as 
specific  as  is  the  immunity  of  an  animal  toward  bacteria. 

Group  reactions  play  a  role  in  chemotherapy  just  as  they  do  in  im- 
munity; the  individual  vaccinated  against  cow  pox  becomes  immune 
toward  small-pox;  the  strain  of  trypanosomes  which  has  become  resistant 
toward  trypan  red  can  no  longer  be  affected  by  trypan  blue. 

Ehrlich's  conception  that  it  is  necessary  for  substances  to  be  taken  up  by  the 
parasites  before  they  can  be  acted  upon  destructively  is  concisely  expressed  by  him: 
"  Corpora  non  agunt  nisi  fixata."  In  terms  of  the  side  chain  theory,  the  protoplasm 
groups  which  have  a  specific  affinity  for  the  chemical  radicles  are  known  as  chemocep- 
tors.  Accordingly,  the  trypanosomes  may  be  said  to  possess  three  distinctly  different 
chemoceptors,  one  directed  against  fuchsin,  one  against  benzopurpurin  and  another 
against  atoxyl. 

While  the  early  chemotherapeutic  studies  with  the  aniline  dyes 
were  mainly  of  theoretical  interest,  the  arsenic  derivatives  soon  claimed 
their  position  of  practical  importance. 

In  1902   Laveran  and   Mesnil    discovered   that  arsenous  acid  could 

16 


242  CHEMOTHERAPY 

destroy  the  trypanosomes  in  the  blood  of  infected  mice;  but  at  the  same 
time  it  acted  as  a  severe  general  poison.  Atoxyl  was  then 
Atoxyl.  introduced  by  Ferd.  Blumenthal  and  was  found  to  be  less 
poisonous  and  just  as  efficient.  Extensive  experiments 
were  undertaken  with  this  drug  and  its  effects  both  in  infected  mice 
and  human  beings  (sleeping  sickness)  carefully  studied.  Most  of  this 
work  was  carried  out  in  the  infected  districts  of  Africa  by  Robert  Koch 
and  his  co-workers.  While  the  trypanocidal  action  of  atoxyl  was  distinct, 
its  effect  was  not  permanent  and  relapses  occurred.  Uhlenhuth  tried 
this  arsenic  compound  in  animal  syphilis,  basing  his  application  upon 
the  apparently  close  relationship  existing  between  the  spirochete  and 
trypanosome  as  pointed  out  by  Schaudinn.  The  results  were  very 
encouraging,  so  that  for  a  time  atoxyl  was  employed  in  the  treatment  of 
human  syphilis  with  beneficial  effect  upon  its  clinical  manifestations.  Its 
use  had  to  be  abandoned,  however,  on  account  of  its  very  severe  asso- 
ciated toxic  actions,  of  which  optic  atrophy  was  the  most  important. 

Up  to  that  time  the  chemical  compositioji  of  atoxyl  had  been  repre- 
sented as  meta-amino-phenylarsenic  acid.  Ehrlich  and  Bertheim  found 
that  this  was  incorrect  and  that  in  reality  it  was  p-amino-phenylarsenic 
acid,  called  by  Ehrlich  arsanilic  acid.  It  was  thus  made  possible  by 
systematic  substitution  to  attain  specifically  active  but  less  poisonous 
products. 

For  a  long  time  too  it  was  unexplained  why  the  trypanocidal  action 
of  atoxyl  was  so  very  marked  in  vivo  and  yet  no  effect  could  be  noticed  in 
vitro.     Ehrlich  presumed  that  when  atoxyl  enters  the  system  it  undergoes 
the  chemical  change  of  reduction;  from  a  quinquivalent  arsenic  combina- 
tion a  trivalent  one  resulted;  and  it  is  this  reduction  product  to  which 
atoxyl  owed  its  activity.     He  proved  this  hypothesis  by  reducing  atoxyl 
in  the  test-tube;  he  thus  obtained  the  phenylarsenious  acid  which  even  in 
the  dilution  of  i  to  i  ,000,000  was  capable  of  destroying  the  trypanosomes, 
whereas  atoxyl  in  the  dilution  of  i  to  100  was  inactive.     "  The  arsenoceptor 
cannot   unite   with   quinquivalent   arsenic  products,  but   requires  ones 
with  trivalent  arsenic;  this  lack  of  saturation  of  the  arsenic  molecule 
is  essential  for  the  chemical  union  with  the  arsenic  receptor"    (Ehrlich). 
The   numerous   trivalent   arsenic   combinations   are   by   no 
Arseno-     means  equally  potent.     Many  are  entirely  unable  to  destroy 
phenyl-     the  protozoa,  others  are  slightly  active,  while  only  few  show 
glycm.      a  distinct  specific  action.     Belonging  to  the  last  class  is  arseno- 
phenylglycin  which  contains  an  acetic-acid  radical.     This  com- 
pound is  peculiar  in  that  it  can  attack  trypanosomes  which  have  become 
resistant  to  atoxyl.     If,  however,  these  "  atoxyl-f ast "  trypanosomes  are 
treated  for  a  long  time  with  arsenophenylglycin,  they  became  resistant 
also  to  the  latter  compound.     According  to  Ehrlich's  explanation  for  this 


VARIOUS   ARSENIC   COMPOUNDS  243 

phenomenon,  the  trypanosomes  possess  in  addition  a  chemoceptor  with  an 
affinity  toward  acetic  acid,  and  therefore  any  strain  which  has  become 
resistant  toward  arsenophenylglycin  has  become  not  only  arsenic  fast  but 
has  become  immune  to  all  combinations  which  like  arsenophenylglycin 
contain  an  acetic  acid  radicle. 

The  action  of  an  arsenical  compound  depends  upon  its  being  anchored 
not  only  by  means  of  one  of  its  groups  but  by  many  groups,  "Just  as  a 
butterfly  that  is  to  be  mounted  must  be  fixed  by  more  than  one  attach- 
ment. First  a  needle  must  be  passed  through  its  body  and  then  successive 
fixation  through  the  animal's  wings  is  necessary."  In  this  sense  Ehrlich 
speaks  of  primary  and  secondary  haptophores.  In  arsenophenylglycin 
the  primary  anchoring  group  is  the  receptor  of  the  acetic  acid  radical 
which  delivers  the  arsenic  element  to  the  cell.  Whereas  for  the  try- 
panosomes it  is  this  acid  group  which  plays  the  main  role,  for  the  spiro- 
chete,  it  is  the  hydroxyl  group,  placed  in  the  para-position  of  the  trivalent 
arsenic  molecule  (arsenophenol). 

As  =  As 


\/  \/ 

OH      OH 

Arsenophenol. 

By  the  introduction  of  different  elements  or  combinations  into  the 
above  formula  (as  iodin  or  an  amido  group)  the  toxic  action  of  the  com- 
pound can  be  further  diminished,  whereas  the  spirillocidal  property  is 
greatly  enhanced.  In  this  manner,  Ehrlich  and  Bertheim  developed  the 
preparation  606  (salvarsan)  which  is  the  dihydrochloride  of  dioxy- 
diamidoarsenobenzol. 


As    =    As 


NH 


NH, 


OH        OH 


As    =••    As 


C1HNH; 


NH2C1H 


OH        OH 


Dioxydiamidoarsenobenzol.  Dioxydiamido-arseno-benzene  dihydrochlor- 

ide (Salvarsan). 

Dioxydiamidoarsenobenzol  is  a  canary  yellow  powder  undergoing  oxidation  very 
readily  so  that  it  is  kept  in  small  vacuum  tubes.  It  is  insoluble  in  water  but  soluble 
if  sodium  hydroxide  is  added.  The  hydrochloric  acid  salt,  salvarsan,  is  soluble  in 
water  giving  a  clear  yellowish  solution.  It  is  acid  in  reaction  and  absorbed  by  the 
system  with  difficulty  (Hata).  On  the  addition  of  sodium  hydrate  first  a  monochloride 
and  then  a  neutral  salt  in  the  form  of  a  flocculent  precipitate  results.  More  of  the  hy- 
droxide redissolves,  the  precipitate  giving  a  clear  solution  of  the  alkali  salt;  its  reaction 
is  strongly  alkaline.  It  makes  no  difference  whether  the  dioxydiamidoarsenobenzol 
is  dissolved  in  sodium  hydrate  or  whether  the  hydrochloric  acid  salt  is  made  alkaline 


244 


CHEMOTHERAPY 


by  adding  the  hydroxide:  in  both  instances  the  sodium  salt  of  dioxydiamidoarseno- 
benzol  is  formed. 

As    =    As 


fH2 


ONa      ONa 

The  toxicity  of  salvarsan  is  comparatively  mild.     The  following  table  of  Hata  shows 
this  in  detail. 


Animal 

Mode  of  application 

Dose  tolerated 

Mouse  

Subcutaneous  

i  :  300  pro  20  g. 

Mouse  

Intravenous.  . 

i  :  3<?o  Dro  20  er 

Rat  

Subcutaneous  . 

o  2       pro     kg 

Hen  

Intramuscular 

o  2^     oro     k.2 

Hen., 

Intravenous 

o  08     pro     ksr 

Rabbit  

Intravenous  .  . 

o  i       pro     kg 

Rabbit  .. 

Subcutaneous  . 

o  i  ^     Dro     ksr 

Salvarsan  has  no  effect  upon  spirochetes  in  the  test-tube.  In  vivo,  however,  Hata 
found  a  single  injection  of  a  relatively  small  dose  sufficient  to  effect  a  complete  cure  in 
mice  infected  by  the  spirillum  of  relapsing  fever.  The  results  are  seen  from  the  fol- 
lowing table. 

CURATIVE  EFFECT  OF  DIOXYDIAMIDOARSENOBENZOL. 

Complete  cure  obtained  after. 


Dose 

i  Injection 

2  Injections 

3  Injections 

600 

100% 

700 

100% 

800 

100% 

IOOO 

75% 

100% 

100% 

1500 

18% 

75% 

100% 

2OOO 

16% 

66% 

100% 

3000 

o% 

o% 

33% 

Salvarsan  is  even  more  active  in  hen  spirillosis;  here  Hata  found  0.0035  gm-  Pro 
kilogram  to  be  a  curative  dose. 

Syphilis  in  rabbits  requires  o.oi  to  0.015  gm.  pro  kilogram  by  intravenous  injection 
for  curative  action. 

After  these  very  favorable  results  were  repeatedly  obtained  in  the  lower 
animals,  salvarsan  was  used  in  man.  A  very  large  number  of  cases  has 
now  been  treated  and  important  and  definite  conclusions  have  been 
reached. 


SALVARSAN    TREATMENT   OF    SYPHILIS  245 

1.  The  toxicity  of  salvarsan  for  the  human  being  either  normal  or 
infected  is  very  slight,  provided  that  the  solution  be  properly  prepared 
and   injected   intravenously.     The   therapeutic   doses   are  non-poisonous. 

Isolated  examples  of  idiosyncrasy  against  salvarsan  may  be  met  with,  but  this  is 
possible  with  any  of  the  more  powerful  drugs.  Such  idiosyncrasy  is  always  more 
liable  after  repeated  administration  (hypersusceptibility). 

In  animals  it  has  been  found  that  a  coexisting  bacterial  infection  may  greatly  raise 
the  poisonous  action  of  salvarsan.  One  should  therefore  guard  against  injecting  a 
syphilitic  who  is  in  addition  suffering  from  an  acute  infection  as  an  influenza. 

2.  Salvarsan  acts  as  a  curative  agent  in  all  varieties  of  spirochete  inva- 
sion.    The  most  favorable  reports  have  been  obtained  in  Febris  recurrens 
(Iversen,  Bitter  and  Dreyer)  and  Frambcesia  tropica  (Koch  and  Flu). 
These  diseases  may  be  completely  cured  by  a  single  injection  of  a  suffi- 
ciently  large   dose.     This   realizes   Ehrlich's   ideal:    "Therapia   magna 
Sterilisans." 

Its  use  in  syphilis  is  also  of  undoubted  value;  it  is  one  of  the 
Syphilis,  most  efficient  antisyphilitic  agents,  and  as  a  rule,  influences 

favorably  the  different  manifestations  of  the  various  stages 
of  lues.  Its  action  is  all  the  more  marked  in  those  individuals  in 
whom  mercurials  seem  to  be  ineffective  (lues  maligna)  or  in  those  having  an 
idiosyncrasy  to  mercury.  One  should  remember,  however,  that  the  radical 
cure  of  syphilis  and  the  disappearance  of  all  the  visible  clinical  manifes- 
tations are  two  entirely  different  considerations.  They  are  by  no  means 
identical,  as  has  been  definitely  shown  by  the  complement  fixation  test 
for  syphilis.  If  a  positive  Wassermann  reaction  is  to  be  taken  as  an 
evidence  of  active  lues  requiring  treatment,  then  it  must  be  granted  that 
some  stages  of  luetic  infections  cannot  be  entirely  eradicated  by  salvarsan 
alone,  even  by  the  intravenous  application.  A  complete  cure  by  one  or 
several  salvarsan  injections  can  be  looked  for  only  in  the  first  or  early 
secondary  stage  of  the  specific  infection,  especially  if  the  Wassermann 
reaction  has  not  yet  become  positive  or  is  only  weakly  so.  Even  here 
ultimate  cure  is  more  certain  if  instead  of  salvarsan  alone,  mercurial 
treatment  is  added  (gray  oil,  calomel,  inunctions).  In  the  other  stages 
there  seems  to  be  no  question  any  longer  but  that  one  must  depend  upon 
this  combined  therapy  for  lasting  and  ideal  results.  Especially  does 
this  pertain  to  those  cases  of  lues  asymptomatica  where  the  only  evidence 
of  a  still  existing  old  infection  is  a  positive  Wassermann  test.  To  make 
the  reaction  negative  in  these  cases  is  no  easy  task.  A  single  intravenous 
injection  of  salvarsan  (0.3  to  0.6  gm.)  never  brings  this  about;  neither 
is  it  accomplished  if  a  few  injections  of  sublimate  or  mercury  salicylate 
are  added.  In  the  early  era  of  the  new  therapy,  the  author  obtained 
much  better  results  in  such  conditions  by  oft  repeated  intramuscular 


246  CHEMOTHERAPY 

injections  in  large  doses  0.6  to  i.o  gm.  per  injection  than  by  the  intravenous 
method.  He  had  to  abandon  this,  however,  on  account  of  the  danger 
from  the  large  quantities  of  arsenic  thus  administered. 

In  order  to  change  such  positive  reactions  it  is  sometimes  necessary 
to  give  as  many  as  four  or  five  intravenous  salvarsan  injections  (0.6  gm.) 
within  a  period  of  three  to  four  weeks,  and  at  the  same  time  undertake 
mercurial  inunctions  or  injections;  then  one  or  two  additional  salvarsan 
injections  (0.6  gm.)  and  finally  large  doses  of  iodides  extended  over  a 
long  period  of  time.  After  a  rest  of  two  months  the  entire  procedure 
may  have  to  be  repeated.  Naturally  this  plan  of  treatment  can  be 
varied  in  many  ways.  It  is  not  advisable,  however,  to  continue  with 
salvarsan  too  long.  It  is  borne  well,  but  its  effect  ceases.  Whether 
the  spirochetes  acquire  a  resistance  toward  arsenic  has  not  been  definitely 
proven,  although  animal  experiments  of  Ehrlich  and  Hata  favor  this 
view. 

The  value  of  salvarsan  in  para-syphilitic  diseases  has  to  a  degree  been 
misjudged  on  account  of  the  early  sensational  communications  of  Alt 
who  claimed  to  have  cured  general  paralysis.  Corroborative  evidence 
by  others  has  been  lacking.  The  treatment  of  cerebrospinal  lues  on  the 
other  hand  is  frequently  attended  by  remarkably  good  results,  and  when 
one  considers  how  closely  this  affection  may  simulate  tabes,  tabo-paral- 
ysis,  or  paresis,  it  is  appreciated  with  what  care  the  diagnosis  of  true 
paralysis  should  be  made.  Here,  while  occasionally  a  certain  symptom 
complex  may  be  improved  there  is  not  sufficient  evidence  to  claim  a 
cure  by  even  'this  latest  most  remarkable  antisyphilitic  agent. 

Salvarsan  has  been  of  service  also  in  other  than  spirochete  infections. 
Most  trustworthy  are  the  results  in  tertian  malaria  (Iversen  and  Werner) 
furunculosis  orientalis  (Nicolle  and  Flu)  and  bilharziosis  ( Johannides) . 
In  anthrax  and  scarlet  fever  the  general  experience  as  yet  allows  of  no 
definite  conclusion.  How  great  a  help  salvarsan  as  an  arsenic  compound 
will  be  in  the  blood  diseases  is  still  to  be  determined. 

The  effects  upon  certain  animal  diseases,  African  glanders  (lymph- 
angitis epizootica),  and  the  spirillosis  of  fowls,  have  been  very  encouraging. 

3.  Harmful  results  from  salvarsan  have  been  described  by  various 
investigators.  As  a  general  rule  no  deleterious  effects  are  observed 
with  the  intravenous  injections  of  properly  prepared  solutions.  The 
intramuscular  and  subcutaneous  injections  were  frequently  attended 
by  severe  local  reactions  which  at  times  led  to  skin  and  muscle  necroses. 
Also  toxic  exanthemata  simulating  scarlet  fever  were  met  with.  Blindness, 
such  as  has  been  ascribed  to  atoxyl  or  arsacetin,  has  not  been  reported. 
On  the  other  hand,  fever,  vomiting  and  diarrhea  often  occur  but  in  most 
instances  are  probably  due  to  bacterial  contamination  of  the  injected 
solution.  Still,  the  sudden  breaking  up  of  the  large  number  of  spirochetes 


NEURO-RECURRENCES   IN    SYPHILIS 


247 


and  the  consequent  liberation  of  their  toxins  can  bring  about  similar 
phenomena.     Usually  all  these  symptoms  pass  off  promptly. 

Somewhat  less  transient  are  affections  of  different  nerves,  which  have 
been  observed  in  cases  of  recent  syphilis  a  short  time  after  injections 
of  salvarsan.  At  first  there  was  some  doubt  whether  these  injuries  were 
due  to  the  action  of  the  new  remedy  or  to  the  original  specific  infection. 
In  most  instances,  they  have  proved  to  be  of  luetic  origin  and  have 
disappeared  under  further  specific  treatment.  Thus  one  dealt  not  with 
a  "  neuro tropic "  action  of  the  salvarsan,  but  with  the  so-called  "neuro 
rezidive"  or  neuro-recurrences  of  syphilis.  Benario  has  demonstrated  by 
the  following  careful  statistics  that  such  " rezidive"  appear  almost  as 
frequently  in  the  early  stages  of  the  infection  when  treated  by  mercury 
alone  as  when  salvarsan  is  employed. 

"NEURO  REZIDIVE"  WITH  SALVARSAN.     194  CASES. 


Right 

Left 

Both  sides 

Unknown 

Total 

Percentage 

Optic 

18 

1  1 

27 

8 

64 

20    I 

Oculomotor  

c 

Q 

2 

•2 

IQ 

8.6 

Trochlear  ...     . 

A 

I 

C 

2    3 

Trigeminus  

2 

•2 
6 

I 

6 

2.  7 

Abducens  

6 

2 

2 

•2 

1-2 

e.o 

Facial  . 

16 

16 

1.6 

16  1 

Acoustic 

IQ 

27 

27 

A 

77 

•3  r    Q 

4 

77 

70 

68 

62 

,0 

• 

22O 

' 

' NEURO  REZIDIVE"  WITH  MERCURY.     SAME  CLASS  OF  CASES  AND  DURING 
SAME  INTERVAL.     122  CASES. 


Right 

Left 

Both  sides 

Unknown 

Total 

Percentage 

Optic. 

10 

g 

77 

2C  .  I 

Oculomotor  

6 

•2 

o 

6 

1C 

i.i  .'5 

Troclilear  

Trigeminus  . 

i 

I 

O.  7 

Abducens  

2 

i 

7. 

2.3 

Facial  

14 

7 

o 

3O 

23.0 

Acoustic  

12 

J7 

J7 

47 

30.8 

Olfactory.... 

I 

I 

o.  7 

Hypoglossal  

I 

o.  7 

These  paralyses  are  due  to  isolated  spirochetes  lying  inaccessible  in 
the  nerve  fibers.     Thus,  probably,  they  escape  the  action  of  the  injected 


248  CHEMOTHERAPY 

antisyphilitic  agents  and  remain  unmolested  only  to  multiply  and  cause 
a  focal  disease  of  the  nerve. 

A  few  reports  of  severer  complications  such  as  meningomyelitis,  or 
death,  due  to  salvarsan  may  be  found  in  the  literature.  Upon  careful 
review  these  instances  reveal  some  responsible  factor  other  than  salvarsan 
alone.  In  this  connection  a  possible  independent  bacterial  infection 
existing  at  the  time  of  the  salvarsan  injection  (as  a  grippe  or  cold)  is 
especially  to  be  emphasized. 

4.  As  centra-indications  for  the  use   of    this   medicament,    Ehrlich 
mentions  more  serious  disturbances  of  the  cardio-vascular  system,  more 
advanced  degeneration  of  the  central  nervous  system,  fetid  bronchitis  as 
well  as  cachexias,  unless  these  be  a  direct  consequence  of  syphilis.     Each 
case,  however,  should  be  considered  by  itself  and  not  as  belonging  to  one  of 
the  above  wide  groups;  thus  while  a  mild  valvular  defect  is  a  cardiac  dis- 
turbance it  does  not  per  se  necessarily  centra-indicate  treatment;  only 
severe  decompensation  should  be  the  inhibitory  factor.     The  same  may 
be  said  of  aneurysms  and  renal  disturbances.     In  incipient  tabes,  in 
early  paralysis  and  epilepsy  of  syphilitic  origin  a  prospect  of  success  can  be 
held  out,  provided  the  treatment  is  commenced  immediately  after  the 
very  earliest  appearance  of  symptoms.     The  doses  of  salvarsan  employed 
here  should  be  smaller,  because  the  susceptibility  is  greater.     In  syphilis 
of    the    liver    the    author   has    observed    rather   poor   results.     Others 
report  the  contrary.    Luetic  infections  of  the  eye,  even  beginning  optic 
atrophy,  offer  no  centra-indication.     In  general,  tuberculosis  is  no  danger 
signal. 

5.  The  mode  of  application  of  salvarsan  is  of  paramount  importance. 
It  may  be  administered  intravenously,  intramuscularly  or  subcutaneously. 

It  can  be  prepared  in  an  alkaline  or  acid  solution,  as  a  neutral  sus- 
pension or  in  an  oil  emulsion.  At  present  the  method  of  choice  is  the 
intravenous  injection  of  an  alkaline  solution. 

The  advantages  of  this  procedure  are  the  absence  of  any  pain,  complete 
resorption  of  the  entire  quantity  injected,  very  rapid  action,  almost  no 
by-effects.  The  only  disadvantage  is  that  the  salvarsan  is  excreted  very 
rapidly  so  that  in  about  four  days  it  has  almost  entirely  left  the  system. 

The  Technique  of  the  Intravenous  Salvarsan  Injection. 

(a)  Preparation  of  the  Alkaline  Solution. — About  30  to  40  c.cm.  of 
0.85  per  cent,  sterile  salt  solution  are  measured  in  a  narrow-necked, 
graduated  glass-stoppered  sterile  cylinder  of  300  c.cm.  capacity,  containing 
about  50  sterile  glass  beads.  (In  the  official  instructions  accompanying  the 
salvarsan,  it  is  advised  to  use  plain  distilled  water  instead.  In  the  author's 
experience  it  has  not  been  found  necessary  to  make  matters  somewhat  com- 
plicated by  using  distilled  water  here  and  further  on  salt  solution.  He 


PREPARATION   OF    SALVARSAN    SOLUTION  249 

uses  either  0.85  per  cent,  salt  solution  or  distilled  water  throughout  the 
preparation  with  excellent  results.  The  0.5  per  cent,  saline  solution  as 
recommended  by  many  instead  of  the  0.85  per  cent,  is  also  a  super- 
fluous refinement.  It  is  however  absolutely  necessary  that  the  sodium 
chloride  is  chemically  pure  and  the  water  used  for  making  up  the  saline 
solution  be  freshly  distilled.  Then  too  in  the  sterilization  of  the  salt  solu- 
tion the  latter  should  not  simply  be  brought  to  the  boiling  point,  but  be 
kept  at  this,  preferably  in  the  moist  heat  sterilizer,  for  a  prolonged  period 
of  time.)  The  salvarsan,  e.g.,  0.6  gm.  is  added.  Upon  vigorous  shaking 
the  substance  goes  into  solution.  It  is  imperative  that  the  salvarsan  be 
completely  dissolved  and  that  no  gelatinous  drop-like  particles  remain. 
Fifteen  per  cent,  caustic  soda  solution  is  now  added  in  accordance  with 
the  following  table : 

0.2  salvarsan  (0.456  gm.)  requires  0.38  c.cm.  15%  Sod.  Hydrate  =8  drops. 
0.3  salvarsan  (0.654  gm.)  requires  0.54  c.cm.  15%  Sod.  Hydrate  =  12  drops. 
0.4  salvarsan  (0.872  gm.)  requires  0.76  c.cm.  15%  Sod.  Hydrate  =15-16  drops. 
0.5  salvarsan  (1.09    gm.)  requires  0.95  c.cm.  15%  Sod.  Hydrate  =19-20  drops. 
0.6  salvarsan  (1.307  gm.)  requires  1.14  c.cm.  15%  Sod.  Hydrate  =23-24  drops. 

(Citron  uses  a  little  more  of  the  alkali;  for  every  o.i  salvarsan,  approxi- 
mately 0.2  c.cm.  sodium  hydrate  solution  is  taken.) 

A  yellow  precipitate  at  once  begins  to  form  and  gradually  increases 
until  a  fine  suspension  ("neutral  suspension")  results;  finally  .  when 
sufficient  of  the  alkali  has  been  added  the  precipitate  redissolves  giv- 
ing a  perfectly  clear  solution.  Its  reaction  is  strongly  alkaline  and 
would  if  injected  in  this  concentrated  quantity  be  sufficient  to  destroy 
the  erythrocytes  and  injure  the  vessel  wall.  Warm  salt  solution  is  there- 
fore added  up  to  250  or  300  c.cm.  If  less  than  0.6  gm.  salvarsan  and 
1.2  c.cm.  sodium  hydrate  have  been  employed,  a  proportionately  smaller 
quantity  of  saline  is  required.  Should  this  solution  not  be  quite  clear 
or  become  slightly  turbid  after  a  few  minutes,  a  few  more  drops  of 
caustic  soda  solution  should  be  added,  a  drop  at  a  time  and  waiting  two 
or  three  minutes  after  each  drop  to  see  if  this  quantity  suffices  to  clear 
the  solution.  Thus  prepared,  it  should  be  used  at  once  and  not 
allowed  to  deteriorate  by  standing,  as  the  oxidation  products  of  sal- 
varsan are  highly  toxic. 

(b)  The  Technique  of  the  Intravenous  Infusion. — The  technique  of  the 
intravenous  injection  of  salvarsan  and  the  instruments  employed  vary 
with  almost  every  physician  who  administers  it.  Each  one  has  his 
"  own  method  and  instrument. "  This  is  hardly  necessary.  The  author's 
technique  is  exceedingly  simple  and  fully  satisfactory. 

The  instrument  consists  of  a  long  narrow  round  graduated  funnel 
of  about  300  c.cm.  capacity;  to  it  is  attached  a  rubber  tube  interrupted 
near  its  lower  end  by  one  or  two  small  pieces  of  glass  tubing  so  as  to 


250  CHEMOTHERAPY 

allow  of  inspection  of  the  fluid  before  it  enters  the  vein;  to  the  rubber 
tubing  is  connected  the  Strauss  canula  or  Schreiber  curved  needle. 
The  whole  is  sterilized  by  boiling  and  then  washed  by  sterile  salt  solution 
flowing  through  it. 


FIG.  28.— First  stage  in  the  intravenous  injection  of  salvarsan.     Needle    being    inserted 
into  distended  vein  and  saline  solution  flowing  into  the  vessel. 

The  steps  in  the  actual  injection  are  as  follows: 

1.  The  patient's  forearm  near  the  median  vein  is  disinfected  (alcohol, 
iodin,  etc.). 

2.  An  assistant  compresses  the  upper  part  of  the  arm  of  the  patient 


INTRAVENOUS   ADMINISTRATION   OF    SALVARSAN 


251 


to  make  the  veins  more  prominent;  this  can  be  further  increased  by  having 
the  patient  make  a  fist. 

3.  The  glass  funnel  filled  with  about  20  to  30  c.cm.  of  normal  saline 
solution  is  supported  at  about  the  level  of  the  patient's  head.  The  air 
is  forced  out  of  the  tubing. 


FIG.  29. — Second  stage  in  the  intravenous  injection  of   salvarsan.     Funnel  lowered,  blood 
flowing  from  vein  into  rubber  tubing  and  can  be  seen  in  the  glass-connection  tube. 

4.  The  operator  inserts  the  needle,  from  which  the  warm  saline  is 
flowing,  into  one  of  the  veins  of  the  elbow  (Fig.  28) ;  care  should  be  exer- 
cised that  the  needle  does  not  penetrate  the  posterior  wall  of  the  vein. 


252  CHEMOTHERAPY 

5.  As  soon  as  the  puncture  has  been  made  the  nurse  lowers  the  funnel 
(Fig.  29);  if  conditions  are  satisfactory  the  saline  in  the  glass  tubing 
near  the  needle  will  become  blood  tinged. 


FIG.  30.— Third  stage  in  the  intravenous  injection  of  salvarsan.     Salvarsan  solution  being 
added  to  the  saline  solution  in  the  funnel. 

6.  All  pressure  upon  the  upper  arm  is  quickly  released,  the  funnel  is 
again  raised  and  the  salt  solution  flows  into  the  vein.     This  should  not 
be  painful  and  no  infiltration  should  form. 

7.  Only  then  is  the  salvarsan  solution  added  to  the  saline  (Fig.  30) 
and  the  funnel  kept  at  the  same  high  level.     (It  is  best  to  have  the 


INTRAMUSCULAR   ADMINISTRATION   OF    SALVARSAN  253 

prepared  salvarsan  solution  transferred  from  the  original  cylinder  with 
the  glass  beads  into  another  empty  one,  so  that  when  the  solution  is  now 
poured  off  into  the  funnel  no  glass  beads  can  fall  into  the  latter.) 

8.  The  infusion  is  stopped  when  the  funnel  empties,  thus  not  making 
use  of  the  fluid  that  remains  in  the  rubber  tubing;  if  also  this  quantity 
is  desired  more  salt  solution  can  be  added  to  the  funnel. 

9.  On  completion,  the  needle  is  removed  by  a  quick  motion. 

The  infusion  should  be  absolutely  painless.  If  it  is  not,  or  if  an  infiltra- 
tion beneath  the  skin  forms,  the  needle  should  at  once  be  removed  and  the 
entire  procedure  repeated  using  another  vein. 

The  dose  for  adults  is  0.6  gm.,  for  children  0.2  to  0.3  gm.,  and  for 
infants  0.02  to  0.05  to  o.i  gm.  Cachectic  or  very  weak  individuals 
should  also  receive  smaller  doses. 

There  are  numerous  modifications  in  the  technique;  one  may  fill  the 
glass  funnel  with  the  prepared  salvarsan  solution  right  at  the  beginning 
but  have  the  rubber  tube  clamped.  The  apparatus  is  fixed  high  up  to  a 
stand.  The  needle  is  detached  from  the  rubber  tube  and  inserted  into 
the  vein;  when  a  distinct  stream  of  blood  escapes,  showing  that  the  needle 
is  within  the  vein,  the  clamp  is  removed  and  the  rubber  tubing  with 
its  escaping  salvarsan  solution  attached  to  the  needle.  Or,  when  the 
needle  is  in  the  vessel  one  may  force  some  salt  solution  with  a  syringe 
through  the  needle  into  the  vein  and  notice  whether  any  infiltration  occurs, 
before  the  salvarsan  solution  is  allowed  to  flow.  Or,  a  three  directioned 
stop-cock  may  be  employed  with  this  object. 

There  is  also  apparatus  with  double  funnels,  one  for  salt  solution 
the  other  for  the  salvarsan;  the  two  are  joined  below  by  their  rubber 
tubings  into  a  single  outlet;  this  allows  of  the  infusion  of  saline  first,  to 
discover  whether  the  vein  has  been  properly  punctured. 

The  Intramuscular  and  Subcutaneous  Injection. 

These  methods  of  administration  have  to  a  great  degree  been  dis- 
continued on  account  of  the  accompanying  severe  local  reaction  and  pain. 
It  is,  also  uncertain  how  much  of  the  salvarsan  thus  injected  is  actually 
absorbed.  Simple  aqueous  (acid)  solutions  of  salvarsan,  10  per  cent., 
alkaline  solutions  with  5  c.cm.  of  fluid,  neutral  emulsions,  and  oily  sus- 
pensions have  been  employed. 

To  prepare  the  neutral  emulsion,  the  salvarsan  powder,  e.g.,  0.5  gm. 
is  placed  in  a  sterile  porcelain  dish  and  triturated  carefully  with  3  to  4 
c.cm.  of  salt  solution.  The  caustic-soda  solution  is  added  drop  by  drop 
(7  to  9)  until  on  testing  with  litmus  paper  the  reaction  is  exactly  neutral. 
If  need  be  a  drop  of  dilute  hydrochloric  acid  may  be  used.  Salt  solution 
or  freshly  distilled  water  is  added  up  to  8  c.cm. 


254  CHEMOTHERAPY 

The  oily  suspension  may  be  made  by  simply  triturating  the  salvarsan  in 
fatty  oils  i  :  10;  Ol.  Amygdal.  dulc,  OL  Sesami,  Ol.  Olivae,  Paraffin,  lodipin. 

The  subcutaneous  injection  is  made  between  the  shoulder-blades  at  the 
sides  of  the  vertebral  column  and  in  a  downward  direction.  It  is  best 
however  to  discard  this  method  entirely. 

The  intramuscular  injection  is  made  in  the  upper  outer  quadrant  of 
the  gluteal  region;  it  should  be  given  deeply  and  very  slowly,  so  as  not  to 
injure  the  muscles.  The  sciatic  nerve  must  be  carefully  avoided.  This 
method  is  chosen  when  the  intravenous  application  is  impossible  (small 
veins,  excessive  fat)  or  when  the  injections  cannot  be  repeated  and  a 
prolonged  action  is  essential.  A  favorite  plan  is  to  give  one  to  two 
intravenous  injections  followed  by  one  to  two  intramuscular  ones. 

Owing  to  the  difficulty  with  which  the  salvarsan  solution  is 
Neosal-      prepared,  and  its  marked  irritating  local  effect,  Ehrlich  pro- 

varsan.  duced  a  modification  of  this  substance  eliminating  these  two 
characteristics.  This  new  chemical  agent,  neosalvarsan ,  is  a 
condensation  product  of  formaldehyde  sulphoxylate  of  sodium  (CH2- 
(OH)O.SO.Na)  and  salvarsan.  Its  composition  is  dioxydiamidoarseno- 
benzol-monomethane  sulphinate  of  sodium.  It  consists  of  a  yellowish 
powder  of  peculiar  odor  and  dissolves  very  easily  in  water,  with  a  com- 
pletely neutral  reaction  resulting. 

The  average  single  dose  of  neosalvarsan  is  half  again  that  of  salvarsan 
as  it  contains  only  66  per  cent,  salvarsan.  On  account  of  its  non-irritat- 
ing properties,  it  is  very  readily  administered  by  intramuscular  injec- 
tion. Subcutaneous  injection  must  be  avoided,  owing  to  the  danger  of 
infiltration. 

The  preparation  of  the  new  solution  is  exceedingly  simple.  The 
powder  dissolves  in  freshly  distilled  water  with  hardly  any  shaking.  A 
0.4  per  cent,  saline  solution  may  be  used  instead  of  the  plain  water, 
provided  it  is  made  from  chemically  pure  sodium  chloride  and  freshly 
distilled  water.  As  with  salvarsan,  solutions  of  neosalvarsan  must  be 
injected  immediately  after  their  preparation.  The  temperature  of  the 
injected  fluid  should  not  rise  above  20-22°  C.  (68  to  71.6°  F.).  Warm- 
ing the  liquid  must  be  avoided. 

For  intravenous  injection  25  c.cm.  of  distilled  water  or  saline  are  re- 
quired for  each  0.15  gram  neosalvarsan;  but  if  desired  it  may  be  admin- 
istered in  much  more  concentrated  solution  as  it  is  by  the  intramuscular 
method;  for  example,  0.6-1.5  gm.  in  10-15  c.cm.  of  fluid.  This  quantity 
can  be  injected  by  means  of  a  15  c.cm.  glass  syringe,  thus  eliminating 
the  use  of  large  or  complicated  apparatus.  Especially  in  children  is  this 
of  advantage. 

The  injections  of  neosalvarsan  are  without  a  doubt  better  borne  than 
a  corresponding  dose  of  salvarsan.  On  this  account  the  new  remedy 


CHEMOTHERAPY    OF    TUMORS  255 

may  be  given  four  or  five  times  at  one  or  two  day  intervals.  Natu- 
rally such  treatment  will  depend  entirely  upon  the  stage  of  the  disease, 
its  clinical  manifestations  and  the  physical  condition  of  the  patient. 

As  for  the  comparative  value  of  salvarsan  and  neosalvarsan,  statis- 
tics are  still  too  few.  Thus  far  it  seems  that  while  the  immediate  effect 
cf  neosalvarsan  upon  active  luetic  lesions  is  similar  to  that  of  salvarsan, 
its  ultimate  effect  upon  the  Wassermann  reaction  and  the  final  eradi- 
cation of  the  syphilitic  infection  is  not  as  striking. 

While  chemotherapy  has  already  made  great  progress  in  protozoon 
infections,  its  importance  in  bacterial  diseases  is  still  very  slight.  The 
drawback  lies  in  the  injury  to  the  normal  tissues  which  occurs  simultane- 
ously with  the  attack  upon  the  pathogenic  bacteria  by  the  chemical  agents. 
Most  favorable  results  in  this  field  have  been  obtained  in  typhoid  fever 
with  Xylene,  and  in  pneumococcus  infections  with  quinine  derivatives 
(Morgenroth).  The  latter  experiments  especially  offer  a  pro  raising  pros- 
pect for  successful  therapy  in  the  human  being. 

Far  more  interesting  and  remarkable   are   the   experiences 

of  A.  von  Wassermann  and  his  co-workers  M.  Wassermann 

iemo-     ancj  Kevsser  wjtn  the  chemotherapy  in  malignant  tumors. 

In  a  mixture  of  tumor  cells  with  sodium  tellurate  and  sodium 
lumors. 

selenate,  it  was  noticed  that  a  certain  affinity  existed  between 
the  cells  and  this  chemical,  in  that  the  metal  was  found 
precipitated  within  the  cell  around  the  nucleus.  This  was  confirmed  in 
living  mice  by  injecting  solutions  of  these  salts  directly  into  the  tumors. 
Thereupon  it  was  shown  that  softening  and  liquefaction  of  the  tumors 
occurred.  Instead  of  the  hard  masses,  a  cyst-like  structure  resulted 
which  usually  ruptured  and  evacuated  a  brownish  semifluid  material. 
This  was  sterile  and  had  a  strong  odor  of  selenium  and  tellurium.  The 
cyst  finally  healed. 

The  next  problem  consisted  in  obtaining  selenium  and  tellurium 
compounds  which  could  be  injected  into  the  circulation  and  thence  reach 
selectively  all  the  carcinoma  cells,  v.  Wassermann  placed  great  stress 
upon  the  " building  of  rails  which  would  reach  the  tumor  and  by  which 
the  .selenium  could  traveJ."  After  painstaking  experiments  involving 
hundreds  of  preparations  he  finally  utilized  eosin  with  its  exceptional 
diffusing  power  as  the  rails  by  which  to  run  the  selenium  into  the  system 
and  send  it  straight  to  the  cancer  cells.  Such  a  chemical  agent  employed 
to  transport  another  substance  to  particular  cells  or  organs  Wassermann 
named  "Cytotrochin." 

"If  three  consecutive  daily  intravenous  injections  of  the  eosin  selenium 
compound  are  given  in  2.5  gm.  doses  for  15  gms.  mice,  a  distinct  softening 
and  elasticity  of  th,e  tumor  are  noticed  on  the  fourth  day;  on  the  fifth  day 
a  fourth  injection  of  the  same  dose  is  given,  after  which  there  is  no  longer 


256  CHEMOTHERAPY 

the  feeling  of  a  solid  tumor  but  rather  that  of  a  fluctuating  cyst  in  which 
small  movable  tumor  particles  can  be  discovered.  After  the  fifth  in- 
jection on  the  seventh  day,  this  soft  mass  becomes  smaller,  the  capsule 
becomes  lax,  and  the  configuration  of  a  circumscribed  tumor  can  no  longer 
be  distinguished,  but  only  a  long  edematous  cord  can  be  felt.  Usually 
as  a  result  of  the  sixth  injection,  in  favorable  cases,  the  absorption  and 
diminution  proceed  so  that  one  gets  the  feeling  of  an  empty  sac.  In  case 
no  intercurrent  disease  occurs,  the  animal  is  cured  in  about  10  days,  with 
a  disappearance  of  all  remnants  of  the  tumor." 

The  intravenous  injection  of  mice  requires  experience.     The  substances 
Intravenous  are  injected  into  the  caudal  vein.     The  mouse  is  put  into  a  special  re- 
Injection     tainer  and  the  cover  of  the  trap  is  closed,  leaving  the  tail  alone  outside, 
in  Mice.     The  mouse  is  fixed  firmly  by  grasping  the  tip  of  the  tail  between  the  left 
thumb  and  index  finger  and  holding  the  tail  fully  extended.     The  caudal 
vein,  which  is  already  somewhat  congested  by  the  pressure  of  the  cover  of  the  retainer 
upon  the  root  of  the  tail,  is  made  more  prominent  by  gentle  exposure  to  heat  in  the 
form  of  a  small  electric  bulb  repeatedly  passed  over  and  close  to  the  skin.     After  a 
little  while  the  superficial  epithelial  cells  become  injured  by  the  heat,  so  that  they 
can  be  removed  by  gentle  scraping  with  a  sharp  scalpel.    This  exposes  the  vein.    The 
successive  inoculations  should  begin  at  the  tip  of  the  tail  and  gradually  approach  the 
root.     A  very  fine  needle  is  essential.     If  the  tip  of  the  tail  becomes  necrotic,  it  should 
be  simply  cut  off. 

The  exceedingly  instructive  observations  of  v.  Wassermann 

Conclusion,  are  at  present  mainly  of  scientific  character.     It  would  be 

erroneous   at   once   to   extend   such   application   to   human 

therapy.     Still,  one  cannot  fail  to  see  in  this  work  the  promise  of  greater 

things  in  the  future.     The  important  principle  that  it  is  possible  to  have 

chemical  substances  pass  from  the  blood  vessels  and  specifically  attack 

tumor  cells  ^has  been  definitely  established;  and  thus  it  seems  merely  a 

question  of  time  before  the  right  step  is  taken  toward  the  solution  of 

one  of  the  greatest  of  human  problems. 


Plate  1, 


Fig.  1.    Positive  v.  Pirquet  Reaction 

(Original  drawing) 


Fig.  2.    Ophthalmo-reaction 

(Original  drawing) 


a)  Control  eye 


b)  Reaction  of  1°— 2°  grade 


INDEX  OF  SUBJECTS 


Abrin,  95 
Acne  Vaccine,  211 
Actinocongestin,  222 
Actinomyces,  196 
African  glanders,  246 

Agglutination  reaction,  differentiation  of  bac- 
terial species  by,  106 
group  agglutination,  no 
partial  agglutionation,  no 
in  cholera  infections,  113 
in  dysentery,  114 

in  epidemic  cerebrospinal  meningitis,  1 14 
in  glanders,  115 
in  Malta  fever,  1 14 
in  paratyphoid  fever,  113 
in  pest,  114 

in  tuberculosis,  114,  115 
in  typhoid  fever,  106,  113 
Agglutinating  sera,  production  of,  109 

preservation  of,  109 

Agglutination  tests,  technique  of,  106-109 
Agglutinins,  against  red  blood  cells,  115;  see 

44  hemagglutinins." 
bacterial,  105-115 
action  of,  105 
biological  structure  of,  160 
definition  of,  105 
diagnostic  value  of,  106 
group  agglutinins,  no 
partial  agglutinins,  in 
preservation  of,  109 
production  of  in  sera  of  animals,  109 
specificity  of,  105-106 
Agglutinogen,  no,  112 
Agglutinoids,  112 
Agglutinophore  group,  112,  160 
Aggressins,  36-44,  61,  232 
aqueous,  39 
artificial,  38,  232 
natural,  36-38 
serous,  39 

Albumin  differentiation,  124-130,  155,  193- 
195;  See  under  " precipitin, "  "pro- 
teid" 
Aleuronat  solution  for  injection  to  produce 

peritoneal  exudates,  138,  197 
Alexin,  133,  152 

Allergic,  224;  see  "Anaphylaxis" 
Amboceptors,  133,  152,  153,  232,  233 
Ananaphylaxis,  224 
Anaphylaxis,  221-230 

Arthus  phenomenon  in,  222 
classical  picture  in  guinea  pig,  223 
due  to  proteids,  222 

to  actino  congestion,  222 
factors  governing  occurrence,  223 
forms  of,  221 

17  257 


Anaphylaxis,  in  dog,  227 

in  guinea  pig,  227-228 

in  hay  fever,  229 

in  man,  223,  226-227 

in  rabbit,  227-228 

in  tuberculosis,  161,  162,  221 

passive,  224 

prevention  of,  methods  for,  224 

relation  to  peptone  poisoning,  228 

serum  sickness,  223,  226,  227 

Theobald  Smith  phenomenon,  222-223 

theories  of,  224-225,  228 
Anaphylatoxin,  225-226 

in  tuberculosis,  161,  221 
Anemia,  pernicious,  100 
Animal  sepsis,  varieties,  22 
Ankylostoma,  170 
Anthrax,  26,  153,  172,  246 
Antiaggressin,  43,  232 
Antiamboceptors,  143,  233 
Antianaphylaxis,  224 
Antibodies,  3,  4,  5,  31,  167,  171,  221 
Antibody  production,  local,  64,  95 
Anticomplements,  155 
Antiendotoxin,  224,  225,  231 
Antiferments,  101-104 

Antigen,  21,  no,  156,  157,  158,  171,  173,  174. 
See  under  "complement  fixation  in 
syphilis." 

Antigenophile  group,  154 
Antihemotoxin,  92-94 
Antikutine,  163 
Antileucocyte  ferment,  102 
Antilysin,  92-94 
Antiserum,  173;  see  under  "serum  therapy 

in— 

Antistaphylolysin,  92-94 
Antitoxin  unit,  80 

Antitoxins,  4,  77-101,  160.  see  under  "diph- 
theria" 

Antitrypsin,  102-104 
Antituberculin,  46, 155, 156, 157, 158, 160, 162 

as  guide  to  prognosis,  163 
Aortitis,  167 
Arachnolysin,  96 
Arsenous  acid,  241 
Arsanilic  acid,  242 
Arsenophenylglycin,  242,  243 
Arthus  phenomenon,  222 
Ascarides,  170 
Atoxyl,  240-242 
"Atoxyl  fast,"  242 
Autoinoculation,  201 

in  tuberculosis,  201-203 
Autumn  catarrh,  229-230 

B 

Bacilli  emulsion  (Koch),  63 
Bacillus  neoformans,  Doyen,  212 


258 


INDEX    OF    SUBJECTS 


Bacterial  destruction  by  heat,  32 

Bacterial  emulsion  in  opsonic  determination, 

205,  206 

Bacterial  extract,  36-44,  173,  234 
Bacterial  filter,  16,  18 
Bacterial  immunization,  31 
Bacterial  precipitin,  121-124;  see  under  "pre- 

cipitin" 

Bactericidal  plate  method,  141-143 
Bacteriolysin,  34,  231 
Bacteriolysis,  131-143,  232 

demonstration  by  Pfeiffer's  method,  131, 

134-140 

by  bactericidal  plate  method,  141-143 
in  vitro,  132 

explanation  of,  133 

necessity  of  complement  for,  132,  133 

phenomenon  of,  131,  133 

relation  to  complement  fixation,  153 

specificity  of ,  131,  133,  134 
Bacteriotropins,  34,  197,  231,  233 

differentiation  from  opsonins,  199 

Neuf eld's  method  of  estimation,  213-215 
Bail's  classification  of  bacteria,  33 
Basedow's  disease,  102 
Basic  silk  dyes,  240 
Bee  posion,  96,  100 
B£raneck's  tuberculin,  63 
Bilharziosis,  246 

Biological  mercurial  therapy,  168-169 
Blood  cells,  washing  of,  98,  145,  147 
Blood  pressure,  fall  in  diphtheria,  84 
Blood  relationship,  125-126 
Blood  removal,  13 

by  wet  cups,  13 
from  vein,  13 
Blood  transfusions,  116 
tests  in,  117,  118 
Bothriocephalus  latus,  100 
Botulism  toxin,  88,  89 
Bovine  tuberculin,  71 
Bovo vaccine,  30,  71 
Brieger's  cachexia  (carcinoma)  reaction,  102 


Cachexia  reaction,  102 
Canula  for  obtaining  blood,  13 
Capillary  pipettes,  136,  203,  204 
Carcinoma,  206-220 

chemotherapy  of,  255-256 

immunity  toward,  216 

serum  diagnosis  of,  102,  206-220 

transplantation  of,  216 
Casein,  103 

Castellani's  test,  in,  112 
Centrifuge  rules,  14 

Cerebro-spinal  lues,  salvarsan  therapy,  246 
Chamberland  filter,  17 
Chemotherapy,  240-256 

group  reaction,  241 

in  bilharziosis,  246 

in  furunculosis  orientalis,  246 

in  malaria,  246 

in  malignant  tumors,  255,  256 

in  pneumonia,  255 

in  syphilis,  245-255 

in  trypanosomiasis,  240,  241 

in  typhoid  fever,  255 

principles  0^240,  241 


Chicken  cholera,  26,  36,  41 
Cholera  extract,  44 

serum,  105,  106-108,  131,  132,  143 
vibrios,  32,  44,  85,   105-115,   134,   135, 

138,  140,  143 
Cholesterin,  89,  99 
Classification  of  bacteria,  23 
Cobra  hemolysis,  97-100 
immunity,  99,  100 
toxin,  96,  97-100 
Coccidiosis,  22 

Coli  bacilli,  2,  44,  107,  172,  205,  207,  211 
Colubrides  poison,  96 
Complement,  171,  174,  200,  231-232 
definition,  132 

importance  of,  in  bacteriolysis,  132-133 
multiplicity  of,  152 
preparation  of,  148,  174 
structure  of,  ,134 
titration  of,  150,  151 
Complement  deviation  (Neisser-Wechsberg), 

i43,  232 
Complement  fixation   or   binding,   152-170, 

171-190 

antigen  in,  171,  173 
antiserum  in,  171,  173 
controls,  174,  175 
definition,  152 
demonstration   of   respective  antigens 

by,  155,  X58,  i77 
antibodies  by,  156,  174-176 
of  antituberculin,  156,  157,  158,  160 
method  for  serum  diagnosis,  153 
principles  of,  152,  153,  154,  158 
reaction   with   exudates   and   transu- 

dates,  173 
relation  of,  to  bacteriolysis,  153,  155 

to  precipitation,  155 
specificity  of,  153,  154 
technique  of,  method  of  Bordet 

Gengou,  171-190 
theories  for  phenomenon,  156 
Complement  fixation  in  diseases  caused  by 

animal  parasites,  170,  190 
in  echinococcus  disease,  170,  190 
in  epidemic  meningitis,  191 
in  gonococcus  infections,  191-192 
in  proteid  differentiation,  193-195 
in  scarlet  fever,  236-237 
Complement  Fixation  in  syphilis,   163-170, 

178-190 
antigens  in,  165 
nature  of,  166 

lippid  extracts,  186,  187,  188 
guinea  pig's  heart,  187,  188,  189 
extract  of  spirochetes,  166 
syphilitic  liver,  166,  182,  185 
preparation  of,  178,  179 
titration  of,  178,  179,  185 
development  of  reaction,  163 
diseases  of  nervous  system,  164 
effect  of  mercurial  treatment,  164,  167, 

168 

of  salvarsan  therapy,  164 
general  principles  of,  152,  153,  154,  158 
guide  to  prognosis,  169 
in  lues  asymptomatica,  165,  167 
in  lues  maligna,  169 
in  mothers  of  syphilitic  children,  159 
in  wet  nurses,  169 


INDEX    OF    SUBJECTS 


259 


Complement  Fixation,  other  diseases   giving 
the  reaction,  166,  185 
modifications  in  technique,  182-190 
Bauer's  method,  189 
Citron's  method,  179,  184,  185,  186 
Noguchi's  method,  187 
Meier's  method,  182-184 
positive  reaction  means  "active"  lues, 

167,  185,  245 

results  in  the  various  stages,  165,  167 
rules  for  its  occurrence,  164 
technique,  179-182 
with  cerebro-spinal  fluid,  164 
Complement  fixation  in   tuberculosis,    155- 

163,  191 

antige    in,  171,  173 
demonstration  of  antituberculin,  155, 

J56,  15? 
theory,  156 
with  urine,  163,  174 
Complement  fixation  in  typhoid  fever,  172, 

192-193 

principle  of,  152,  153,  154 
preparation  of  antigen,  171,  173,  192 
Complementoids,  147 
Complementophile  group,  133,  160 
Conjunctiva  reaction,  52,  54,  57,  58 

in  hay  fever,  229 
Control  tests,  value,  5,  6,  174 
Cow-pox,  25 
Crotin,  96 
Cutaneous  reaction  in  tuberculosis,  49,  50,  55, 

58 

Cytase,  152,  198 
Cytolysin,  151 
Cytophile  group,  133,  154 
Cyto  toxin,  151 
Cytotrochin,  225 

D 

Death  of  bacteria,  32 
Desiccator  for  drying  serum,  15 
Deviation  of  complement,  143 
Dilutions,  18 

preparation  of,  18,  19,  20  . 
Diphtheria,  74-84 
Diphtheria  serum  or  antitoxin,  77-84 

preparation  of,  77,  78 

prophylactic  application,  84 

standardization,  79-82 

therapeutic  application,  82-84 
Diphtheria  toxin,  action  in  animals,  75-76 

estimating  strength  of,  76 
,      obtaining  of,  74-75 
Donath  Landsteiner's  test,  100-101  j 
Dysentery  antitoxin,  90-92 

bacilli,  32,  44,  90-92,  114,  143 

serum,  90-92,  114,  143 


Echinococcus,  170,  igo 
Ehrlich's  experiment,  100 

side  chain  theory,  112,  158-160 
Endocarditis  maligna,  113 
Endotoxin,  85,  138,  224,  225 
Eosin  selenium,  255,  256 
Ergophore  group,  112,  147 
Erysipelas,  236 


Erythrocytes;  see  "blood  cells" 
Exudate,  36,  37,  136,  173 
Extracts  of  bacteria,  36-44 

production  of,  138 

removal  of,  12,  136 

staining  of,  136 


Fatty  acids,  100 
Febris  recurrens,  166 
Ferments,  101-104 

biological  structure  of,  160 
Fever  in  tuberculin  reaction,  161 
Picker's  diagnosticum,  107 
Filters,  bacterial,  16 
Filtration,  16 
Focal  reaction,  46,  64 
Food-stuff  substitution,  125,  127,  128,  129 
Forensic  serum  differentiation,  125,  129 
Fornet's  ring  test,  123 
Fowl  plague,  36 
Frambesia,  166 
Friedberger's  position,  n,  136 
"Frigo"  for  preserving  serum,  16,  148 
Fuchsin,  240 
"Fuchsinfast,"  241 
Functionating  radicle  of  cell  (biological),  158, 

159 

Furunculosis,  200 
orientalis,  246 


Glanders,  115 

diagnosis  with  mallein,  58 
Glycogen,  155 
Gonococcus  vaccine,  211 
Group  agglutination,  110-112 
Group  reactions,  122 
Guinea  pig  sepsis,  21 

H 

Hamburger's  local  reaction,  47 
Haptine,  160 

Haptophore  group,  112,  147,  160 
Hay  fever,  229-230 

diagnosis  of,  229 

serum  therapy,  229-230 
Helminthiasis,  170 
Hemagglutinins,  115-119 

in  transfusions,  116 
Hemoglobinuria,  100-101 
Hemolysis  due  to  cobra  toxin,  97-100 
Hemolysins,  140-151,  171 

characteristics  of  hemolysins,  144 

estimation  of  strength,  146-149 

immune  hemolysins,  144-151 

normal  hemolysins,  144 

phenomena  of  hemolysis,  144 

preservation  of,  146 

production  of  by  immunization,  144-145 
Hemolytic  system,  147-149 
Hemorrhagin,  97 
Hemotoxin,  86,  87,  96-100 
Hen  spirillosis,  244 
Hog  cholera,  4,  31,  113,  135 
Hydrophobia,  26-30 
Hypersusceptibility;  see  "anaphylaxis  " 


260 


INDEX    OF    SUBJECTS 


Immune  bodies,  133 

after   typhoid    prophylactic    injections, 

34 

Immune  hemolysin,  144-151 
Immunity,  3 

definition,  221 

absolute  and  relative,  4 

active,  3 

acquired,  216 

antiaggressin,  40 

antitoxic,  100 

attained,  4 

chicken  cholera,  41 

conception  of,  3 
diphtheria,  77-84 

against  hog-cholera,  4,  113 

against  pure  parasites,  41 

in  lues,  167,  168 

against  snake  poison,  100 
swine  pest,  37,  40,  41,  42 

bactericidal,  3,  40 

cellular,  3 

continued,  4 

general,  4 

"histogene,"  3 

local,  4,  63,  95 

natural,  4 

"pan  immunity,"  216 

to  tumors,  216 

partial,  63,  no 

passive,  4,  231,  239 

tissue,  3 

transitory,  4 

tuberculin,  162 
Immunization,  active  principle  of,  21,  77 

technique,  22,  77 

with  aggressin,  40-45 

with  dead  virus,  31-34 

with  erythrocytes,  145 

with  living  virus,  24-31 

with  toxins,  77-83 
Inactivation,  132,  174 
Incubation  period,  27,  75,  78,  86,  223 

in  anaphylaxis,  223 
Injection,  technique,  9-12,  37 

intracardial,  10,  83 

intracerebral,  86 

intramuscular,  83 

intraneurol,  88 

intraperitoneal,  83 

intraspinal,  234-236 

intravenous,  9,  10 

subcutaneous,  12,  83 

subdural,  88 

Intracutaneous  tuberculin  reaction,  51 
Isoagglutinins  or  Isohemagglutinins,  116-118 
Isoprecipitins,  126 
Isohemolysins,  116-118 


Jennerian  immunization,  26 
Jequirity  seed,  95 

K 

Kaolin,  228 

Kidney  tuberculosis,  67 


Killing  of  bacteria,  31,  32 
Klausner's  reaction,  124 
Kolles'  flasks,  38 


Laboratory  equipment,  7-9 
Law  of  multiple  proportions,  85 
Lecithin,  89,  97,  98,  99 

tuberculin,  73 

hemotoxins,  97-100 
Leprosy,  166 

treatment  with  nastin,  72 
Leucoantifermantin,  102 
Leucocidin,  151 
Leucocytes,  4,  36 

obtaining  of,  182,  213,  214;  see  also  under 
"opsonic  index,"  "bacteriotropin" 
Lilliputian  filter,  17 
Limes  death,  80,  81 

zero,  80,  8 1 

Lipoids;  see  "  lecithin,"  90,  and  "  cholesterin  " 
Loeffler's  serum  plates,  102 
Loop  standard,  8,  19 
Lues;  see  "complement  fixation  in  syphilis" 

asymptomatica,  165,  245 
Lupus,  67 
Lyssa,  26-30,  60 


Macrocytase,  152,  198 
Macrophage,  196,  197 
Malaria,  113,  166 

salvarsan  in,  246 
Mallein,  57,  58 
Malta  fever,  114,  212 
Measles,  41,  124,  185 
Meiostagmine  reaction,  218-220 
Mendelian  law  for  agglutinins,  116 
Meningitis,  epidemic,  114 

serum  diagnosis,  175-177 

serum  therapy,  234-236 
Meningococci,  32,  172 
Meningococcic  serum,  113,  234,  236 

titration  t>f ,  175-177 
Mercurial  therapy  (biological)  168,  169 

combined  with  salvarsan,  245,  246 
Metchnikoff's  resistance  test,  138,  196-197 
Microcytase,  152,  198 
Mouse  typhoid  bacilli,  31,  70,  113 
Much  and  Holzmann  reaction,  99 
Multipartial  sera,  in,  112 


X 


Nastin,  72 

Negative  phase,  78,  200 

Neosalvarsan,  254-255 

Nephrotoxin,  151 

Neurorecurrences,  247-248  ("neurorezidive") 

Neurotoxin,  86,  87,  97,  99,  151 

Neutral  red  for  vital  stain,  198 

New  tuberculin,  62-63,  69-71 

Nonagglutinable  strains  of  bacteria,  112,  113 

Nonbinding  doses,  156-158,  174 

Normal  bacteriolysins,  138 

Normal  curative  serum,  79 

Normal  hemolysin,  144 


INDEX    OF    SUBJECTS 


26l 


Normal  loop,  10,  19,  20 
Normal  toxins,  79 

O 

Obtaining  blood,  12-14 

Ointment  reaction,  Moro,  50 

Oleic  acid,  98-99 

Ophthalmo  reaction,  52-54,  160 

Opsonic  index,  199-209,  212,  213 
definition,  199 

diagnostic  importance  of,  201 
increase  by  immunization,  200-201 
relation  between  index  and  clinical  con- 
dition of  patient,  201,  212,  213 
technique  for  determination  of,  203-209 
variations  by  auto-inoculation,  201,  202 

Opsonins,  198-213 

Opsonizer,  205 

Organotrope,  240 

Original  tuberculin,  old,  61 

Ozena  bacilli,  123 


Paradoxical  reaction,  240 
Paralysis,  progressive,  164,  165,  167 
Parasites,  23,  41,  232 

half,  23,  41,  232 

total,  23,  41 
Parasitotrope,  240 1 
Parasyphilitic  infections,  246 
Paratyphoid  bacilli,  30,  110-113,  140,  143 
Paroxysmal  hemoglobinuria,  100,  101 
Partial  agglutinins,  no 
Partial  aggressins,  62 
Partial  immunization,  63,  no 
Pathogenicity,  23 

Peptone  poisoning  in  anaphylaxis,  228 
Peritoneal  exudate,  136 

method  for  removal,  12,  136 
Pernicious  anemia,  100 
Pest,  114,  153,  172,  238 

sera,  114,  131,  238 
Pfeiffer's  phenomenon,  131 

technique  and  practical  application,  134- 
140 

in  cholera,  139 
Phagocytosis,  139,  196-215 

cells  engaged  in  phagocytosis,  196-197 

definition,  196 

demonstration  of,  197,  198 

object  of  phagocytosis,  196-197 

during  artificial  immunity,  196-213 
'  during  natural  immunity,  4 
Phagolysis,  197 
Phrynolysin,  96 
Phytotoxin,  95-96 
Pipettes  for  removal  of  peritoneal  exudates, 

136 

v.  Pirquet's  reaction,  48-50,  55 
Pneumococci,  32,  199,  237-238 
Pneumococcic  sera,  119,  237-238     '    ., 
Pneumonia,  102 

chemotherapy  of,  255 

bacilli,  123 
Pollantin,  230 
Pollen  poison,  229-230 
Polyvalent  sera,  in,  232 
Porges  reaction,  123 


Positive  phase,  78,  200 
Preservation  of  serum,  15 

in  "Frigo,"  16 
Precipitation,  120-130 
Precipitate  in  precipitin  reactions,  130 
Precipitins,  120-130 

biological  structure  of,  120,  160 
phenomenon  of  precipitation,  120 
bacterial,  121-124 

clinical  value  of  in  typhoid,  122,  123 

in  syphilis,  123,  124 
Fornet's  ring  test,  123 
group  reactions  of,  122 
Klausner's  reaction,  124 
Porges'  reaction,  123 
precipitating  antisera  for,  production 

of,  121 

precipitinogens,  121 
proteid,  124-130 
action  of,  124,  125 
determining  nature  of  meat,  by,  125, 

127,  128,  129 
differentiation  of  proteids  by  Uhlen- 

huth's  method,  127-128 
distinguishing  blood  of  animal  species, 

125,  126 

production  of  precipitating  antisera, 

126,  127 

Prognostic  employment  of  autoinoculation, 
201 

of  the  lues  reaction,  167,  169,  185 

of  the  tuberculin  reaction,  58-59 
Prophylactic  inoculations  in  cholera,  35 

hog  cholera,  31 

hydrophobia,  28 

small-pox,  27 

typhoid,  33-35,  44  . 
Prophylaxis  in  diphtheria,  84 

in  dysentery,  90-92 

against  hay  fever,  230 

in  pest,  238 

in  tetanus,  88 

in  ulcus  corneae,  237 

Proteid  differentiation  by  complement  fixa- 
tion, 193-195 

by  precipitins,  124-130 

Proteid  anaphylaxis,  221-230;  see  "anaphy- 
laxis" 

Pseudo-agglutination,  108 
Psycho-reaction,  99 
Puerperal  sepsis,  237 
Pukal  filter,  17 

R 

Rabbit  sepsis,  21 
Rabies,  26-30 

simultaneous  treatment  of,  30 

vaccination  against,  28-30 
Rat  trypanosomiasis,  22,  240,  241 
"Reagine,"  166,  etc. 
Receptors,  133,  147,  151,  158-160,  231,  232- 

233 

Reichel  filter,  17 
Relapsing  fever  in  mice,  salvarsan  therapy, 

244 

Resistance  test  of  Metchnikoff,  138 
Rheumatic  fever,  236 
Rhinoscleroma  bacilli,  123 
Ricin,  95 
Ring  test,  123 


262 


INDEX    OF    SUBJECTS 


Saprophytes,  23 
Salvarsan,  243-255 

chemical  formula  and  properties,   243- 

244 

therapy  in  hen  spirillosis,  244 
therapy  in  relapsing  fever,  244 
therapy  in  syphilis  of  man,  245-255 
therapy  in  other  diseases,  245-246 
Salvarsan  therapy  in  human  syphilis,  245-255 
action,  245,  246 
combined  with  mercurial  therapy,  245, 

246 

contraindications,  248 
harmful  effects,  246,  248 
mode  of  application,  248-255 
intramuscular,  253-254 
intravenous,  248,  249-253 
subcutaneous,  253-254 
neuro-recurrences  in,  247 
value  in  parasyphilitic  diseases,  246 
Scarlet  fever,  124,  166,  173,  185,  246 
Scorpion  poison,  96,  100 
Scrofulous  reaction  to  tuberculin,  50 
Seiden  peptone,  155,  228 
Selenium,  255-256 
Sensitized  bacteria,  70,  143 

advantages  of,  70 
Sensitized  tuberculin,  70-71 
Sepsis,  113,  221,  237 
of  guinea  pigs,  22 
of  rabbits,  21,  23 
Serum,  color,  15 

bacteriolytic,  231 
obtaining  of,  12,  13,  79 
preservation  of,  15,  16,  79 
value  in  therapy,  231-232 
Serum  diagnosis;  see  under  individual  infec- 
tious diseases  and  under  "Comple- 
ment fixation" 
of  malignant  tumors,  216 

antitrypsin  test,  122-124,  217 
Freund-Kaminer  reaction,  217-218 
Meiostagmine  reaction,  218-220 
of  meningitis,  175-177 
of  syphilis,  123,  124.     see  under  "com- 
plement fixation" 
"Serum  fast,"  233 
Serum  sickness,  83,  223,  226-227 
Serum  therapy,  231-239 
in  anthrax,  239 
in  cholera,  239 
in  diphtheria,  83-84 
in  erysipelas,  236,  237 
in  hay  fever,  229-230 
in  meningitis,  234-236 
in  pest,  238 
in  pneumonia,  237 
in  puerperal  fever,  237 
in  rheumatism,  236 
in  scarlet  fever,  236,  237 
in  sepsis,  237 

in  strep tococcic  infections,  236-237 
in  tetanus,  88 

in  tuberculosis,  162,  233,  238 
in  typhoid  fever,  239 
in  ulcus  serpens,  237 
Sessile  receptors,  160,  161 
Side  chain  theory,  158-160 


"Simultaneous  method,"  30,  31,  77,  238 

definition,  31 
Small-pox,  25 

Smith's  phenomenon,  222-223 
Snake  poison,  96-100 

serum,  100 
Specificity,  i,  5,  54,  105,  no,  125,  134 

of  Koch  subcutaneous  reaction,  56 

of  ophthalmo  reaction,  57 

of  v.  Pirquet  reaction,  55 

of  tuberculin  reaction,  54 
Spermatoxin,  151 
Spider  poison,  96 
Spirillosis  of  fowls,  246 
Spirochete  infections,  240 
Standard  loop,  8 
Staphylococci,  31,  44,  92,  199,  200,  205 

vaccine,  209-210 
Staphylohemotoxin,  92-94 
Staphylolysin,  92-94 
Sterilisatio  Magna,  240,  245 

mode  of  action,  240-241 
Strauss  canula,  13 
Street  virus,  27 
Streptococci,  32,  173,  206 

sera,  236-237 

vaccine,  211-238 

Subcutaneous  tuberculin  injections,  45-49,  56 
Substance  sensibilisatrice,    133;   see  "ambo- 

ceptor,"  "bacteriolysin" 
Summation  of  antigen,  156,  157,  174 
Swine  erysipelas,  30 

pest,  36,  40,  41,  42,  43 
Syphilis,  123,  124;  see  "atoxyl,  '    salvarsan, 

"complement  fixation." 
System  of  hemolysis  tests,  147 


Tabes  dorsalis,  164,  165 
Tauruman,  30,  71 
Tebesapin,  72 
Tetanolysin,  86,  87 
Tetanospasmin,  86,  87 
Tetanus,  86,  87 

antitoxin,  88 

cerebral,  86 

sine  tetano,  86 

Thermolabile  substances  in  serum,  15,  16,  133 
Thermoresistant  substances  in  serum,  15,  109, 

i33 

Thyroid,  102 
Titration  of  antigen,  178,  179,  185 

of  bacteriolytic  serum,  137 

of  complement,  150 

of  hemolysin,  146-149 

of  luetic  sera,  179-182 

of  virulence  of  cultures,  34 
Toad  poison,  96 
Toxins,  74-104,  147 

action  of,  74,  75 

definition,  85 

dilution  of,  18 

obtaining  of,  74-75 

titration  of,  76 

of  higher  plants  and  animals,  95-101 
Toxoids,  81,  147 
Toxolipoids,  100 
Toxon,  80 
Toxophore  group,  147,  159 


INDEX    OF    SUBJECTS 


263 


Toxopeptids  in  relation  to  anaphylaxis,  228 

to  tuberculosis,  161 
Transfusion  of  blood,  116-118 
Transplantation  of  tumors,  216 
Transudate,  173 

Traumatic  tuberculin  reaction,  50 
Trichophytin,  58 
Trypan  blue,  240 

red,  240 
Trypanosomiasis,  22,  166,  240-241 

trypanocidal  agents,  240 
Trypsin,  102-104 
Tubercle  bacilli,  60,  61,  62,  172,  196 

emulsion  in  opsonic  determinations,  206 
Tuberculin,  45-73,  155-163,  200,  201-203 
action,  63-64 
as  antigen,  162 
Beraneck's  tuberculin,  63 
bovine  tuberculin,  71-72 
"depot"  reaction,  47 
diagnosis,  45-49 

cutaneous,  v.  Pirquet,  49-50 
explanation  of,  156,  157,  160,  161 
Hamburger's  method,  47 
intracutaneous,  51 
ointment  reaction,  50-51 
ophthalmo  reaction,  52-54,  57,  58 
subcutaneous  reaction,  45-49 
preparations,  62-63,  70 

new  tuberculin,  63,  69-71,  211 
obtaining  of,  45-46 
old  tuberculin,  45-59,  62,  65 
original  old  tuberculin,  62 
vacuum,  62 
watery,  62 
reaction,  46-54 
theories,  of  Citron,  160-161 
of  Wassermann,  156,  157 
therapy,  60-71,  155-162 

antituberculin  production  during  ther- 
apy, 155-162 
contraindications,  68-69 
general  principles,  64-65 
with  bovine  tuberculin,  71-72 
with  new  tuberculin,  69-71 
with  old  tuberculin,  65-69 
with  sensitized  tuberculin,  70-71 
Tuberculosis,    99,    114-115,    233    see   under 

"complement  fixation" 
prognosis  in,  from  local  reaction,  58-59 
sera  for,  99,  114,  115,  238 
treatment  of,  by  Friedmann  vaccine,  30 
in  successive  steps,  66 
with  nastin,  72 
with  tebesapin,  72 
with      tuberculin;     see     "tuberculin 

therapy" 
vaccination  in,  30,  212-213 


Tuberculosis  of  kidney,  67 

Tumors,  malignant;  see  under  "carcinoma" 

Typhoid  bacteria,  32,  106-113,  123,  134,  135, 

171-173,192-193 
immunization  of  rabbit  with,  32 
titration  of  virulence  of  culture,  24 
extract,  44 

Typhoid  fever,  141,  185,  239,  255 
protective  inoculations,  33-35 
serum,  106,  113,  140,  141,  239 
vaccine,  33-35,  44,  211 

U 

•Ulcus  corneae,  238 
Unit  of  antitoxin,  80 

Urine,    complement  fixation   test  for  tuber- 
culosis, 163,  174 
Urticaria,  227,  229 


Vaccination  against  rabies,  26-30 

against  small-pox,  25-26 

against  tuberculosis,  30,  212-213 

against  typhoid,  33-35 
Vaccines  (Wright),  209-213 

dosage,  211-212 

preparation,  209-211 
Vacuum  desiccator,  15,  16 

tuberculin,  62 
Venopuncture,  12,  13 
Viper's  poison,  100 
Virulence  of  bacteria,  24 

method  for  determining,  134 
virulence  of  typhoid,  134 
diminishing  virulence,  232 
increasing  virulence,  135,233 
Virus  fixe,  27 
Vital  staining,  198 

W 

Wall  of  leucocytes,  233 

Wassermann's   reaction;     see    "complement 

fixation" 

Watery  tuberculin,  62 
Weigert's  law,  159 

Wet  cupping  for  obtaining  blood,  13 
Wet  nurse,  examination  for  syphilis,  169 
Whooping  cough  bacilli,  173 
Widal's  test,  106-108 
Wright's  pipettes  and  tubes,  203 

method  for  obtaining  blood,  204 


Zootoxins,  96-100 


INDEX  OF  AUTHORS 


Allessandrini,  99 
Amiradzibi,  218 
Anderson,  87 
Apolant,  216 
Arloing,  114 
Arndt,  93 
Aronson,  236,  239 
Arrhenius,  86 
Arthus,  222,  223,  227 
Ascoli,  218,  219 
Audeoud,  57 
Auer,  227 
Aufrecht,  68 

Bail,  23,  31,  36,  38,  40,  42,  44,  46,  139 

Bamberg,  103 

Bandelier,  48,  69 

Bandi,  238 

Bashford,  216 

Bassenge,  44 

Bauer,  99,  189,  190 

Benario,  247 

v.  Behring,  2,  30,  71,  77,  79,  80,  88,  160,  189, 

190 

Beraneck,  63 
Bergell,  239 
Berger,  50 
Berghaus,  82,  83 
v.  Bergmann,  102,  103 
Bernstein,  220 
Bertheim,  242 
Bertrand,  99 
Besredka,  223,  224,  239 
Biedl,  227,  228 
Bier,  14 
Bitter,  245 
Blaschko,  165 
Blumenthal,  166,  242 
Boas,  168 
Bockenheimer,  88 
Boer,  79 
Bordet,  3,  31,  86,  120,  132,  143,  144,  152, 

153,  154,  171 
Borrel,  86 
Borelli,  165 

Brieger,  44,  102,  139,  187,  217 
Bruck,  64,  92,  93,  155,  156,  164,  165 
Buchner,  133 

Calmette,  52,  88,  99,  100,  238 

Castellani,  in,  113 

Chamberland,  31 

Chantemesse,  239 

Christian,  160 

Citron,  38,  52,  57,  62,  154,  160,  165,  166,  178, 

184,  225,  233 
Coca,  98 
Cohen,  173 
Conradi,  90,  109 


Courmont,  114 
Craig,  1  66 
Czaplewski,  8 

Dagonet,  216 

Denys,  62,  198 

Deutsch,  239 

Deycke,  72 

Diatroptoff,  30 

Doenitz,  83,  86 

Doerr,  90,  91,  223 

Doganoff,  50 

Donath,  100,  101 

Dopter,  91 

Douglas,  198 

Doyen,  212 

Dreyer,  245 

Dunbar,  229 

v.  Dungen,  98,  100,  116,  144 

Durham,  105 

Ehrlich,  2,  79,  80,  81,  82,  85,  87,  95,  100,  132 
144,  147,  152,  158,  198,  240,  242, 
246,  254 

v.  Eisler,  122 

Ellerman,  55 

Eppenstein,  52,  57 

Epstein,  116,  118 

Erlandsen,  55 

van  Ermenghem,  88 

Escherich,  237 

Ferran,  29 

Ficker,  107 

Fleischmann,  165 

Fleming,  190 

Flexner,  90,  97,  234,  235,  236 

Flu,  245 

Foix,  173,  236 

Fornet,  22,  123 

Fournier,  168 

Frankel,  C.,  77 

Franz,  56 

Freeman,  203 

Freund,  217,  218 

Friedberger,  n,  12,  16,  136,  161,  223,  225 

Friedmann,  31 

Friedman,  116 

Friedemann,  223,  225 

Fuld,  103 

Garbat,  178,  187,  192,  239 

Gay,  227 

Gengou,  3,  143,  152,  153,  154 

Ghedini,  170 

Goetsch,  65 

Graff,  218 

Gross,  103 

Gruber,  105,  233 


265 


266 


INDEX    OF    AUTHORS 


Hamburger,  47 
Hartung,  227 
Hata,  243,  244,  246 
Hecht,  189 
Helmann,  58 
Hendersen-Smith,  85 
Herbst,  128 
Heubner,  83 
Hogyes,  29 
Hoke,  44 
Holzmann,  99 
Hiippe,  44 

Issaeff,  136 
Iversen,  245,  246 
Izar,  218 

Jaffe,  142,  143 
Jenner,  3,  25 
Jensen,  216 
Jobling,  234 
Jochmann,  102,  234 
Johannides,  246 
Joseph,  51 

Kaminer,  217,  218 

Kelning,  58 

Kempner,  89 

Keysser,  161,  228,  255 

Kikuchi,  44 

Kitasato,  74,  77,  87 

Kitashima,  77 

Klausner,  124 

Kleine,  115 

Klinkert,  162,  163 

Koch,  R.,  30,  45,  47,  48,  56,  60,  64,  71,  114, 

242 

Kolle,  32,  35,  44,  108,  138,  139 
Koplik,  227 
Korte,  141,  143 
Kossel,  82 
Kramer,  68 
Kraus,  R.,  82,  85,  90,  91,  120,  178,  223,  227, 

228,  234,  239 
Kreibich,  124 
Kruse,  90 
Kuhn,  35 
Kutscher,  114 
Kyes,  97 

Landsteiner,  100,  101,  144,  186,  188 

Laubry,  170 

Laveran,  241 

Leclef,  198 

Ledermann,  165 

Leishman,  33,  198 

Lenhartz,  67 

Lesourd,  154,  192 

Leuchs,  173,  192 

Levaditi,  165,  197 

Lewin,  C.,  216 

Liefmann,  155 

Liepmann,  216 

Lignieres,  50 

Loewenstein,  49,  162 

Lohlein,  199 

Lorenz,  31 

Liidke,  192 

Lustig,  238 


Mac  Fadyen,  239 

Madsen,  78,  79,  83,  86,  89 

Mallein,  173,  236 

Mantoux,  51 

Manwaring,  228 

Maragliano,  62,  238 

Marcus,  102 

Marie,  30,  165 

Markl,  238 

Marmorek,  163,  174,  236,  238 

Martin,  77 

Marx,  82 

Matthews,  213 

Mayer,  44 

McNeil,  191 

Meier,  G.,  123,  165,  182,  183,  184,  186 

Meakins,  191 

Mendel,  51 

Menzer,  236 

Mesnil,  241 

Metallnikoff,  72 

Metschnikoff,  2,  138,  139,  151,  152,  196,  232 

Meyer,  87 

Meyer,  F.,  70,  83,  162,  165,  178,  236,  239 

Meyer,  K.,  102,  103 

Michaelis,  G.,  92,  93 

Micheli,  165 

Miessner,  128 

Mita,  226,  228 

Moller,  68 

Moreschi,  154,  155 

Morgenroth,  10,  13,  16,  83,   144,   147,   152, 

158,  165,  255 
Moro,  50 
Moser,  236 
Much,  99 

Muller,  102,  186,  188,  191 
Munk,  166,  187 

Nakayama,  156,  158,  174 

Negri,  26 

Neisser,  M.,  92,  141-143,  155,  164,  165,  168, 

193 

Netter,  83 

Neufield,  166,  199,  213,  214 
Nichols,  1 66 
Nicolle,  153,  246 
Nocard,  26 
Noguchi,  26,  72,  97,  100,  187,  189 

Obermeyer,  31,  129 
Oppenheim,  191 
Ostertag,  31 
Ottenberg,  116,  118 
Otto,  89,  223 

Pane,  237 

Parvu,  170 

Pasteur,  3,  26,  27,  41 

Petit,  57 

Petruschky,  66 

Pfeiffer,  35,  44,  108,  131,  134-140,  198,  223, 

226,  228 
Phisalix,  99 
Pick,  31,  129 
Pickert,  162 

v.  Pirquet,  48,  52,  55,  223,  224,  226 
Plato,  58 

Plaut,  123,  164,  165 
Porges,  122,  123,  186 


INDEX    OF    AUTHORS 


267 


Portiers,  222 
Potzl,  1 86,  1 88 
Prausnitz,  230 

Rabinowitsch,  158 

Radziewsky,  136 

Ransom,  87 

Remlinger,  26,  30 

Reschad,  72 

Ribbert,  233 

Richet,  222 

Rietschel,  169 

Romer,  51,  82,  95,  237 

Ropke,  48,  70 

Rosculet,  92 

Rosenau,  87,  223 

Rosenblatt,  160 

Rosenthal,  90,  91 

Roux,  26,  31,  74,  77,  82,  86 

Ruppel,  70,  162,  234 

Russel,  34 

Sachs,  H.,  89,  97,  155,  186,  193 

Sahli,  63 

Salimbeni,  238 

Salmon,  31 

Salomonsen,  78,  79 

Salus,  44 

Schaudinn,  242 

Schenck,  57 

Schick,  223,  224,  227 

Schittenhelm,  223,  226 

Schleissner,  173,  236 

Schone,  192 

Schucht,  164,  165 

Schulze,  92,  93 

Schiitze,  125,  164,  165 

Schwartz,  191 

Schwarz,  82 

Sclavo,  239 

Seiffert,  57 

Seligmann,  165 

Shiga,  44,  98 

Simons,  220 

Smith,  31 

Smith,  Theob.,  222,  227 

Sobernheim,  239 

Southardt,  227 

Spengler,  62 

Steinberg,  143 


Steinhardt,  224 
Stenitzer,  239 
Stern,  Marg.,  165,  190 
Stern,  141,  148 
Stertz,  165 
Strauss,  13 
Szaboky,  99 

Takaki,  86 
Tallquist,  100 
Tavel,  238,  239 
Teague,  191 
Teichmann,  59 
Terni,  238 
Todd,  90,  91 
Topfer,  142,  143 
Torrey,  191 
Toussaint,  31 
Traube,  218 
Trebing,  102 
Tschernogubow,  189 
Tschistowitsch,  120 

Uhlenhuth,  9,  n,  125-130,  242 

Vaillard,  84 
Vannod,  191 
Vigano,  218 

Wassermann,  A.,  2,  3,  n,  38,  64,  86,  89,  114, 

125,   155,  156,  161,   164,   165,   178, 

232,  255,  256 

Wassermann,  M.,  228,  255 
Wechsberg,  92,  141-143 
Weichardt,  218,  223,  225,  229 
Weidanz,  189 
Weigert,  159 

Weil,  36,  41,  156,  158,  174 
Weinberg,  170,  190 
Werner,  246 
Widal,  1 06,  154,  192 
Wolff-Eisner,  52,  54,  59,  223,  224,  225,  229, 

231 

Wollstein,  191 
Wright,  3,  32,  33,  44,   108,   198,   199,   200, 

209,  etc. 

Yersin,  74,  238 

Zeuner,  72 
Zupnik,  87 


YC  88631 


360085 


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UNIVERSITY  OF  CALIFORNIA  LIBRARY 


$§i£^li: 


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